Process of producing glutamate derivatives

ABSTRACT

The present invention relates to a process for producing efficiently glutamic acid derivatives (including salts thereof) such as monatin by converting a substituted α-keto acid of formula (1) into a glutamic acid derivative of formula (2) in the presence of an enzyme catalyzing conversion of the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 11/561,665,filed on Nov. 20, 2006, which is a divisional of U.S. Ser. No.10/876,468 (now U.S. Pat. No. 7,297,800), filed on Jun. 28, 2004, whichis a continuation of PCT/JP02/12852, filed on Dec. 9, 2002, which claimspriority to JP 2002-245980, filed on Aug. 26, 2002, JP 2002-095760,filed on Mar. 29, 2002, and JP 2001-396471, filed on Dec. 27, 2001. Theentire contents of these applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process of producing glutamatederivatives using enzyme reactions. Further, the invention relates to aprocess of producing monatin using tryptophan as a starting material.

2. Discussion of the Background 4-(Indol-3-ylmethyl)-4-hydroxy-glutamicacid (3-(1-amino-1,3-dicarboxy-3-hydroxy-butan-4-yl)-indole)(hereinafter referred to as “monatin”) represented by the followingstructural formula (6) is an amino acid contained in the root of a shrubtree (e.g., Schlerochitom ilicifolius) in South Africa. Monatin has alevel of sweetness that is several hundreds-fold greater than that ofsucrose. Therefore, monatin is a particularly promising low-caloriesweetener (see JP-A-64-25757).

4-(Indol-3-ylmethyl)-4-hydroxy-glutamic acid

Monatin has asymmetric carbons at positions 2 and 4. Naturally occurringmonatin has a (2S,4S) steric configuration. In addition to the (2S,4S)steric configuration, the non-naturally occurring stereoisomers may besynthetically produced. It has been determined that all of the otherstereoisomers of monatin have a highly enriched sweetness level comparedto sucrose. Therefore, it is expected that any single monatinstereoisomer or mixture of monatin stereoisomers may be utilized as asweetening agent or as a component for a sweetening agent (e.g., asweetener).

Five examples of monatin production processes have been reported. Thedetails of these processes are as described in the following referencesof the related art.

(1) Specification of U.S. Pat. No. 5,994,559

(2) Tetrahedron Letters, 2001, Vol. 42, No. 39, pp. 6793-6796

(3) Organic Letters, 2000, Vol. 2, No. 19, pp. 2967-2970

(4) Synthetic Communication, 1994, Vol. 24, No. 22, pp. 3197-3211

(5) Synthetic Communication, 1993, Vol. 23, No. 18, pp. 2511-2526

However, each of these processes requires multiple steps. Moreover, anindustrial-scale production process is yet to be established. Thus, ademand exists for the development of a simple, high-yield industrialprocess for producing glutamate derivatives, including monatin andanalogs thereof.

Thus, it is an object of the invention to provide an efficient processof producing glutamate derivatives and salt forms thereof includingmonatin. These compounds are promising as a component for a sweetener.

SUMMARY OF THE INVENTION

To achieve the aforementioned object, the present inventors havesuccessfully developed a production method for a glutamate derivative(including salt forms thereof) of formula (2)

(R¹ and R² in formula (2) have the same meanings as R¹ and R² in thegeneral formula (1)). In this process, the glutamate derivative of thegeneral formula (2) is produced from a substituted α-keto acid offormula (1) in the presence of a catalytic enzyme

(in formula (1), R¹ and R² independently represent a substituentselected from the group consisting of a hydrogen atom, an alkyl grouphaving one to 8 carbon atoms (C1 to C8), a C1 to C8 alkoxyl group, a C2to C9 carboxyalkyl group, an aryl group having up to 20 carbon atoms, anaralkyl group having up to 20 carbon atoms, a heterocyclicring-containing hydrocarbon group, and a hydroxyl group; when one of R¹and R² represents a hydrogen atom, however, the other is not a hydrogenatom, a methyl group or an ethyl group; when one of R¹ and R² representsa hydroxyl group, the other is not a hydrogen atom or a methyl group;when R¹ contains an aromatic ring or a heterocyclic ring, the aromaticring or the heterocyclic ring may be additionally substituted with ahalogen atom, a hydroxyl group, alkyl groups with up to 3 carbon atoms,alkoxyl groups with up to 3 carbon atoms and an amino group), underconditions and for a time suitable to progress the reaction. Based onthe finding, the invention has been achieved.

The process for producing glutamate derivatives in accordance with thepresent invention enables efficient production of monatin represented byformula (6) from 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid(hereinafter referred to as “IHOG”) represented by formula (7), using anenzymatic reaction.

The present inventors have also developed new methods of producingmonatin from tryptophan as a starting material, including the followingreactions 1 to 3. The process of producing the glutamate derivatives ofthe present invention corresponds to reaction 3 in the monatinproduction process resulting from the following reactions 1 to 3. Theproduction route of monatin, including reactions 1 to 3, is shown inreaction scheme (8).

-   -   Reaction 1: preparing indole-3-pyruvic acid from tryptophan in        the presence of an enzyme catalyst.    -   Reaction 2: preparing the precursor keto acid (IHOG) via aldol        condensation between indole-3-pyruvic acid and pyruvic acid (or        oxaloacetic acid).    -   Reaction 3: synthetically preparing monatin by aminating IHOG at        the position 2 in the presence of an enzyme catalyst.

The present invention is described in more detail below.

[1] A process for producing a glutamate derivative, or salt formsthereof, of formula (2)

(R¹ and R² in formula (2) have the same meanings as R¹ and R² in formula(1)). In this process, the glutamate derivative of the general formula(2) is produced from a substituted α-keto acid of formula (1) in thepresence of a catalytic enzyme

(in formula (1), R¹ and R² independently represent a substituentselected from the group consisting of a hydrogen atom, an alkyl grouphaving one to 8 carbon atoms (C1 to C8), a C1 to C8 alkoxyl group, a C2to C9 carboxyalkyl group, an aryl group having up to 20 carbon atoms, anaralkyl group having up to 20 carbon atoms, a heterocyclicring-containing hydrocarbon group, and a hydroxyl group; when one of R¹and R² represents a hydrogen atom, however, the other is not a hydrogenatom, a methyl group or an ethyl group; when one of R¹ and R² representsa hydroxyl group, the other is not a hydrogen atom or a methyl group;when R¹ contains an aromatic ring or a heterocyclic ring, the aromaticring or the heterocyclic ring may be additionally substituted with ahalogen atom, a hydroxyl group, alkyl groups with up to 3 carbon atoms,alkoxyl groups with up to 3 carbon atoms and amino group), underconditions and for a time suitable to progress the reaction.[2] A process for producing a glutamate derivative as described in [1],wherein R¹ represents a phenylmethyl group or a 3-indolylmethyl group,and R² represents a hydroxyl group.[3] A process for producing a glutamate derivative as described in [1]or [2], wherein the enzyme is a dehydrogenase or a transaminase.[4] A process for producing a glutamate derivative as described in [3],wherein the enzyme is a transaminase and the reaction system thereforcontains one or more types of amino acids as amino group donors.[5] A process for producing a glutamate derivative as described in [4],wherein the amino acids are selected from the group consisting ofglutamic acid, aspartic acid, alanine, tryptophan, phenylalanine,isoleucine, leucine, tyrosine, valine, arginine, asparagine, glutamine,methionine, ornithine, serine, cysteine, histidine and lysine.[6] A process for producing a glutamate derivative as described in [3]through [5], wherein the enzyme is a L-amino acid transaminase.[7] A process for producing a glutamate derivative as described in [3]through [5], wherein the enzyme is a D-amino acid transaminase.[8] A process for producing a glutamate derivative as described in [7],wherein the reaction system therefor contains an enzyme that catalyzesthe conversion of a L-amino acid to a D-amino acid.[9] A process for producing a glutamate derivative as described in [6],wherein the L-amino acid transaminase is derived from a microorganismbelonging to a genus selected from the group consisting of Aeromonas,Agrobacterium, Alcaligenes, Beijerinckia, Escherichia, Proteus andMorganella.[10] A process for producing a glutamate derivative as described in [9],wherein the microorganism is selected from the group consisting ofAeromonas hydrophila, Agrobacterium tumefaciens, Alcaligenes faecalis,Beijerinckia indica, Escherichia coli, Proteus rettgeri and Morganellamorganii.[11] A process for producing a glutamate derivative as described in [7]or [8], wherein the D-amino acid transaminase is derived from amicroorganism of the genus Bacillus or Paenibacillus.[12] A process for producing a glutamate derivative as described in[11], wherein the microorganism is selected from the group consisting ofBacillus sphaericus, Bacillus pulvifaciens, Bacillus macerans, Bacilluslentus, Paenibacillus larvae subsp. pulvifaciens and Paenibacillusmacerans.[13] A process for producing a glutamate derivative as described in [1],wherein the enzyme is generated by a microorganism transformed with agene encoding a D-amino acid transaminase.[14] A process for producing a glutamate derivative as described in[13], wherein the microorganism is Escherichia coli.[15] A process for producing a glutamate derivative as described in [13]or [14], wherein the gene encoding a D-amino acid transaminase is fromBacillus sphaericus or Bacillus macerans.[16] A process for producing a glutamate derivative comprising:

[I] producing a substituted α-keto acid of formula (4)

(R in formula (4) has the same meaning as R in formula (3)) in thepresence of an enzyme catalyzing the production of substituted α-ketoacid of formula (4) from a substituted α-keto acid represented byformula (3)

(in formula (3), R represents a substituent selected from the groupconsisting of a C2 to C8 alkyl group, a C1 to C8 alkoxyl group, a C2 toC9 carboxyalkyl group, an aryl group having up to 20 carbon atoms, anaralkyl group having up to 20 carbon atoms, a heterocyclicring-containing hydrocarbon group, and a hydroxyl group; when R containsan aromatic ring or a heterocyclic ring, the aromatic ring or theheterocyclic ring may be additionally substituted with a halogen atom, ahydroxyl group, an alkyl group having up to 3 carbon atoms, an alkoxylgroup having up to 3 carbon atoms and an amino group), and oxaloaceticacid or pyruvic acid, under conditions and for a time suitable toprogress the reaction; and

[II] producing a glutamate derivative, or salt forms thereof,represented by formula (5) in the presence of an enzyme catalyzing thereaction production of glutamate derivative of formula (5)

(R in formula (5) has the same meaning as R in formula (3)) from thesubstituted α-keto acid of formula (4), under conditions and for a timesuitable to progress the reaction.[17] A process for producing a glutamate derivative as described in[16], wherein R represents a phenylmethyl group or a 3-indolylmethylgroup.[18] A process for producing a glutamate derivative as described in [16]or [17], wherein the enzyme catalyzing the reaction in [I] is derivedfrom a microorganism belonging to a genus selected from the groupconsisting of Pseudomonas, Erwinia, Flavobacterium and Xanthomonas.[19] A process for producing a glutamate derivative as described in[18], wherein the microorganism is Pseudomonas taetrolens, Pseudomonascoronafaciens, Pseudomonas desmolytica, Erwinia sp., Flavobacteriumrhenanum or Xanthomonas citri.[20] A process for producing a glutamate derivative as described in[19], wherein the microorganism is Pseudomonas taetrolens ATCC4683 orPseudomonas coronafaciens AJ2791.[21] A process for producing a glutamate derivative as described in [16]or [17], wherein the enzyme catalyzing the reaction in [I] is any of thefollowing proteins:

(a) a protein comprising the amino acid sequence of SEQ ID NO. 2;

(b) a protein comprising an amino acid sequence prepared bysubstitution, deletion, insertion, addition and/or inversion of one orseveral amino acid residues in the amino acid sequence of SEQ ID NO. 2where the protein has aldolase activity;

(c) a protein comprising the amino acid sequence of SEQ ID NO. 3;

(d) a protein comprising an amino acid sequence prepared bysubstitution, deletion, insertion, addition and/or inversion of one orseveral amino acid residues in the amino acid sequence of SEQ ID NO. 3where the protein has aldolase activity.

[22] A process for producing a glutamate derivative described in [16] or[17], wherein the enzyme catalyzing the reaction in [I] is an enzymeobtained from a recombinant where the gene encoding any of the followingproteins is amplified and expressed:

(a) a protein comprising the amino acid sequence of SEQ ID NO. 2;

(b) a protein comprising an amino acid sequence prepared bysubstitution, deletion, insertion, addition and/or inversion of one orseveral amino acid residues in the amino acid sequence of SEQ ID NO. 2having aldolase activity;

(c) a protein comprising the amino acid sequence of SEQ ID NO. 3;

(d) a protein comprising an amino acid sequence prepared bysubstitution, deletion, insertion, addition and/or inversion of one orseveral amino acid residues in the amino acid sequence of SEQ ID NO. 3having the aldolase activity.

[23] A process for producing monatin comprising:

[A] producing indole-3-pyruvic acid in the presence of an enzymecatalyzing the conversion of tryptophan to indole-3-pyruvic acid;

[B] producing 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid fromindole-3-pyruvic acid, and oxaloacetic acid or pyruvic acid;

[C] producing monatin in the presence of an enzyme catalyzing theproduction of monatin from 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaricacid, by allowing 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid.

[24] A process for producing monatin as described in [23], wherein [A]includes reacting tryptophan in the presence of an enzyme catalyzing theconversion of tryptophan to indole-3-pyruvic acid, and treating theresulting reaction solution with any of deaeration treatment, deoxygentreatment and pH adjustment up to pH 2 at maximum to collectindole-3-pyruvic acid.[25] A process for producing monatin as described in [24], wherein thedeaeration treatment or the deoxygen treatment is a process ofsubstituting the whole or a part of the gas contained in the reactionsolution with an inactive gas.[26] A process for producing monatin as described in [25], wherein theinactive gas is selected from the group consisting of nitrogen, argonand helium.[27] A process for producing monatin as described in [24] through [26],wherein the pH is adjusted by adding an acid to the reaction solution,and the process further comprises crystallizing indole-3-pyruvic acidproduced as a consequence of the pH adjustment and collecting theresulting indole-3-pyruvic acid.[28] A process for producing monatin as described in [27], wherein theacid is any of sulfuric acid, hydrochloric acid, nitric acid andphosphoric acid.[29] A process of producing monatin as described in [23] through [28],wherein the enzyme catalyzing the reaction at [A] is derived from amicroorganism having amino acid oxidase activity and catalase activity.[30] A process for producing monatin as described in [23] through [29],wherein the enzyme catalyzing the reaction at [A] is a microorganismbelonging to a genus selected from the group consisting ofAchromobacter, Proteus and Morganella.[31] A process for producing monatin as described in [30], wherein theenzyme is from a microorganism selected from the group consisting ofAchromobacter sp. AJ2425, Proteus rettgeri IFO13501 and Morganellamorganii IFO3168.[32] A process for producing monatin as described in [23], wherein [A]comprises interacting a culture of a microorganism with tryptophan,wherein said microorganism possesses an ability to convert tryptophan toindole-3-pyruvic acid and is a member of a genus selected from the groupconsisting of Achromobacter, Proteus, Morganella, Pseudomonas andNeurospora, and further comprises producing indole-3-pyruvic acid andthen collecting indole-3-pyruvic acid.[33] A process of producing monatin as described in [23] through [32],wherein [B] is performed in the presence of an enzyme catalyst.[34] A process of producing monatin as described in [23] through [32],wherein [B] is a chemical synthetic method.

The above objects highlight certain aspects of the invention. Additionalobjects, aspects and embodiments of the invention are found in thefollowing detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined, all technical and scientific terms usedherein have the same meaning as commonly understood by a skilled artisanin enzymology, biochemistry, cellular biology, molecular biology, andthe medical sciences.

All methods and materials similar or equivalent to those describedherein may be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

The process of producing glutamate derivatives developed by theinventors is for producing glutamate derivatives of formula (2) from asubstituted α-keto acid of formula (1). More particularly, the processof the present invention relates to the production of glutamatederivatives in which an enzyme-catalyzed transamination is performedusing an isolated enzyme or a microorganism generating the enzyme.

Additionally, the process for producing monatin from a starting materialtryptophan developed by the inventors includes the following reactions 1to 3. It should be noted that the process of producing monatin includingthe following reactions 1 to 3 utilizes the process of producingglutamate derivatives in accordance with the invention as reaction 3.

-   -   Reaction 1: synthesis of indole-3-pyruvic acid from tryptophan        in the presence of an enzyme catalyst.    -   Reaction 2: synthesis of the precursor keto acid (IHOG) by aldol        condensation between indole-3-pyruvic acid and pyruvic acid (or        oxaloacetic acid).    -   Reaction 3: synthesis of monatin by aminating IHOG at the        position 2 in the presence of an enzyme catalyst.

Among the reactions 1 to 3, the reactions 1 and 3 are enzymaticreactions. However, the reaction 2 may be carried out by either chemicalsynthesis or enzymatic synthesis, with no specific limitation.

The process for producing monatin in accordance with the invention isnot limited to the process of producing monatin using tryptophan as thestarting material but preferably includes the reaction 3 as an essentialstep among the reactions 1 to 3. In the other words, the invention alsoincludes a process for producing monatin using commercially availableindole-3-pyruvic acid as the starting material via the reactions 2 and3, and a process of producing monatin from the precursor keto acid(IHOG) as the starting material for reaction 3. Thus, the process ofproducing monatin in accordance with the invention includes all of thefollowing processes (a) through (c).

(a) Reactions 1+2+3

(b) Reactions 2+3

(c) Reaction 3 alone

Also in the present invention reaction 2 may be used for the productionof the substituted α-keto acid that may be used as the substrate in theprocess of producing glutamate derivatives in accordance with theinvention in addition to being used for the synthesis of the precursorketo acid (IHOG) of monatin.

As shown in scheme (10), the process of producing a glutamate derivativeof formula (5) via reaction 3, using the substituted α-keto acid offormula (4) as obtained via reaction 2 (Reaction 2+Reaction 3), is alsoincluded in the process of producing glutamate derivatives in accordancewith the invention.

The invention is now described in details with reference to the attacheddrawings, sequentially in the order of [A] Reaction 1, [B] Reaction 2and [C] Reaction 3.

[A] Reaction 1

Reaction 1 shown in scheme (11) is a reaction related to the productionof indole-3-pyruvic acid. Reaction 1 in accordance with the inventioncharacteristically includes a step in which tryptophan is converted toindole-3-pyruvic acid in the presence of a catalytic enzyme. Theresulting reaction solution is subsequently treated with any ofdeaeration treatment, deoxygen treatment or pH adjustment up to no morethan pH 2 to collect indole-3-pyruvic acid.

In the related art, a chemical process of producing indole-3-pyruvicacid was proposed by Giovanna De Luca, et al. and has been disclosed.This process includes a step of reacting tryptophan with pyridinealdehyde in the presence of a base for dehydrating proton acceptors toobtain indole-3-pyruvic acid at a yield of 50 to 62% (see thepublication of Patent Publication (TOKUHYO) No. Sho 62-501912, thepamphlet of International Publication WO 87/00169). According to theprocess of DeLuca et al, the base and pyridine aldehyde are essential,but are very expensive and the resulting the yield is low. Therefore,the production cost is very high, which is problematic.

Additionally, a process for producing indole-3-pyruvic acid at arecovery of 64% was proposed by Politi Vincenzo, et al. The Vincenzoprocess uses a condensation reaction between indole andethyl-3-bromopyruvate ester oxime followed by acid hydrolysis (EP 421946). According to this process, a purification step using silica gel isneeded and the resulting yield is low. Moreover, the raw materials areexpensive. Thus, the process is disadvantageous in terms of its highcost for application to industrial production.

An enzymatic process for producing indole-3-pyruvic acid using atransaminase is also known (see scheme (12) below).

Moreover, reports exist of a process of producing indole-3-pyruvic acidby reacting L-tryptophan (L-Trp) with L-tryptophan transaminase fromCandida maltose to generate indole-3-pyruvic acid from 40 mM L-Trp and80 mM 2-ketoglutaric acid. The product is then purified with an ionexchange resin and the resulting indole-3-pyruvic acid is obtained at ayield of 72% (see East Germany Patent DD 297190 to Bobe Ruediger, etal.).

Another process for producing indole-3-pyruvic acid has been profferedin which L-Trp and 2-ketoglutaric acid react with an aspartatetransaminase to generate indole-3-pyruvic acid. Subsequently thereaction solution is extracted in petroleum ether, and indole-3-pyruvicacid is purified by column chromatographic separation to collect thepurified indole-3-pyruvic acid (see JP-A-59-95894 to Mario Matterazzi,et al.).

However, these processes using transaminase provide a low yield andrequire keto acid such as 2-ketoglutaric acid to serve as an amino groupacceptor in addition to L-Trp. In addition, these processes involve thesecondary production of an amino acid corresponding to the amino groupacceptor at a molar amount equivalent to the molar amount of theproduced indole-3-pyruvic acid. Further, more keto acid is required thanL-Trp in the reaction system to improve the overall yield. As such,residual keto acid still remains even after the completion of reaction.Based on these factors, the collection of intended indole-3-pyruvic acidfrom the reaction solution requires a purification step using ionexchange resins or the like, which involve complicated procedures andhigh cost.

As a process for producing indole-3-pyruvic acid from L-Trp, further, aprocess using L-amino acid oxidase has also been known. Becauseindole-3-pyruvic acid is decomposed to indoleacetic acid (see scheme(14)) with hydrogen peroxide secondarily produced during the tryptophanoxidation by L-amino acid oxidase (see scheme (13)), herein, a processis proposed, including adding catalase to the reaction system todecompose the hydrogen peroxide (see scheme 15) (see the specificationof U.S. Pat. No. 5,002,963; Tetrahedron Letters, 1987, Vol. 28, No. 12,pp. 1277-1280).

Specifically, the process entails employing an enzyme-immobilized columnprepared by immobilizing L-amino acid oxidase from snake venom- andbovine liver-derived catalase on a carrier (e.g., a resin). The solutioncontaining L-Trp is then passed through the column to permit thereaction, the indole-3-pyruvic acid produced on an ion exchange columnis subsequently absorbed, the product is then eluted with methanol anddried to collect the product. According to the process, however, 0.5 gof the starting L-Trp results in only 0.2 g of indole-3-pyruvic acid, ata yield as low as 40%. The enzyme immobilization procedure and thepurification procedure using ion exchange resin are laborious andrequire a step for recovering and recycling the residual L-Trp. Thus,the process is disadvantageously very costly.

Concerning L-amino acid oxidase derived from microorganisms,alternatively, John A. Duerre, et al. detect the oxidation activity ofL-Trp by measuring the activity, including a step of approximatelypurifying the L-amino acid oxidase from Proteus rettgeri and a step ofdetecting oxygen consumption (see Journal of Bacteriology, 1975, Vol.121, No. 2, pp. 656-663). Additionally, Furuyama, et al. confirm thatthe L-phenylalanine oxidase derived from Pseudomonas sp. P-501 interactswith L-Trp by measuring the oxidase activity, including a step ofdetecting oxygen consumption (see Noda Institute for ScientificResearch, Kiyofumi Maruyama, Journal of Biochemistry, 1990, 108, pp.327-333).

In these reports, however, oxidase activity is detected by means ofmeasuring the L-tryptophan consumption, oxygen consumption and theamount of hydrogen peroxide generated during the enzyme reactions, butindole-3-pyruvic acid is never directly assayed. This may be due to thefact that indole-3-pyruvic acid is decomposed to indoleacetic acid byhydrogen peroxide produced via the reaction with amino acid oxidase.Meanwhile, no example of indole-3-pyruvic acid production using amicrobial cell or a treated product from microbial cells exists to date.Therefore, it has been unknown how microorganisms decompose tryptophanor what decomposition products are produced thereby.

Additionally, the process described above using transaminase and theprocess using snake venom-derived L-amino acid oxidase for theproduction of indole-3-pyruvic acid are at low reaction yields andrequire chromatographic separation steps for the recovery ofindole-3-pyruvic acid, due to the presence of by-product keto acid andresidual L-tryptophan in the reaction solution. Thus, the processesrequire very laborious procedures and involve high production costs.

Accordingly, the present inventors have investigated how to provide aprocess of producing indole-3-pyruvic acid in a simple manner and at lowcost. The inventors have discovered that the interaction of amicroorganism having amino acid oxidase and catalase activities withtryptophan may produce indole-3-pyruvic acid. The resultingindole-3-pyruvic acid may then be collected. Particularly, the presentinventors have found that the interaction of the aforementionedmicroorganism with tryptophan may produce indole-3-pyruvic acid,preferably with suppression of the decomposition of the intended productby inactive gas substitution or pH adjustment for the resulting reactionsolution, which may be collected.

In addition to the decomposition of indole-3-pyruvic acid toindoleacetic acid with hydrogen peroxide, the inventors have discoveredthat indole-3-pyruvic acid is attacked with oxygen and the like in thesolution resulting in decomposition products with unknown structures.Accordingly, the solution containing indole-3-pyruvic acid eventuallybecomes colored. Therefore, the present invention provides a method forsolving the problem.

In accordance with the invention, tryptophan is reacted in the presenceof an enzyme catalyzing the conversion of tryptophan to indole-3-pyruvicacid and the resulting solution is treated with any of the followingmethods to facilitate collection of indole-3-pyruvic acid: deaerationtreatment, deoxygen treatment, or pH adjustment up to pH 2 at maximum.

The decomposition or coloring of indole-3-pyruvic acid may progress atits solution state. By the acid addition process, however,indole-3-pyruvic acid crystallizes during the early stage of the step ofcollecting the resulting indole-3-pyruvic acid. Therefore, compared withother purification and treatment steps, the acid addition process mayadvantageously suppress the decomposition and coloring.

Indoleacetic acid as a decomposition product of indole-3-pyruvic acid isnot always readily removed under acidic conditions by directcrystallization. However, the secondary production of indoleacetic acidmay be effectively suppressed via inactive gas substitution. Acombination of crystallization under acidic conditions and inactive gassubstitution may be more highly effective for the high-purity collectionof indole-3-pyruvic acid.

Additionally, an alternate mode for performing reaction 1 in accordancewith the present invention includes interacting a culture of amicroorganism with tryptophan, wherein said microorganism possesses anability to catalyze the conversion of tryptophan to indole-3-pyruvicacid, to produce and collect indole-3-pyruvic acid.

Heretofore, no report has existed about the interaction of a culture ofa microorganism with tryptophan, wherein said microorganism possesses anability to catalyze the conversion of tryptophan to indole-3-pyruvicacid, to produce and collect indole-3-pyruvic acid. Thus, the processprovides a novel and useful process for producing indole-3-pyruvic acidby an enzymatic method.

The mode for performing reaction 1 in accordance with the presentinvention is now sequentially described in reference to (A-1) the enzymefor use in reaction 1 and (A-2) reaction conditions for reaction 1.

(A-1) Enzyme for Use in Reaction 1

The enzyme for use in reaction 1 includes any enzyme having an abilityto convert tryptophan to indole-3-pyruvic acid, with no specificlimitation. As the enzyme for use in reaction 1, enzymes having aminoacid oxidase activity, and enzymes having catalase activity arepreferable.

The “amino acid oxidase activity” for reaction 1 means an activitycatalyzing the reaction shown in scheme (13). Generally, L-amino acidoxidase generates keto acid from the corresponding L-amino acid, whileD-amino acid oxidase generates keto acid from the corresponding D-aminoacid. Specifically, in accordance with the present invention,individually, a microorganism having L-amino acid oxidase activity maybe used when L-tryptophan is used as the raw material, while amicroorganism having D-amino acid oxidase activity may used whenD-tryptophan is used as the raw material. Additionally, the preparationfrom DL-tryptophan is also applicable. When D- and L-amino acid oxidaseis allowed to interact with DL-tryptophan, the intended indole-3-pyruvicacid may be quantitatively produced. When D- or L-amino acid oxidaseinteracts with DL-tryptophan, otherwise, the intended indole-3-pyruvicacid may be produced at a yield of 50%.

Additionally, the “catalase activity” means an activity catalyzing thereaction shown in scheme (15).

The enzyme with amino acid oxidase activity for reaction 1 may beselected by using any of various known methods, such as an assaydetecting oxygen consumption due to the oxidation activity of amino acid(see for one example, Journal of Bacteriology, 1975, Vol. 121, No. 2,pp. 656-663) and a method of measuring hydrogen peroxide generated viathe reaction (see for one example M. Gabler, et al., Enzyme andMicrobial Technology, 2000, 27, pp. 605-611) as well as the method ofdirect measuring indole-3-pyruvic acid produced from tryptophan asdescribed below in accordance with the present invention.

The enzyme having catalase activity for reaction 1 may be selected byusing any of various known methods, including a method of measuring thehydrogen peroxide decrease via the catalase reaction based on the changeof the absorbance at 230 nm to 250 nm, a method of measuring theresidual hydrogen peroxide in the reaction solution with KMnO₄, and amethod of measuring oxygen produced during the reaction with amanometer. As one example, a spectroscopic method of measuring theresidual hydrogen peroxide is listed, which includes a step of oxidizingelectron donors such as o-dianisidine via the peroxidase reaction, asdescribed in M. Gabler, et al., Enzyme and Microbial Technology, 2000,27, pp. 605-611. Using any of these methods, an enzyme having catalaseactivity may be selected.

Furthermore, the enzyme for use in reaction 1 may be selected bydetecting the activity for producing indole-3-pyruvic acid fromtryptophan, according to the method described below in Example 1.

The microorganism generating the enzyme for use in reaction 1 may beselected for example from a genus represented by Achromobacter, Proteus,Morganella, Pseudomonas and Neurospora. The microorganism is preferablya microorganism having amino acid oxidase activity and catalaseactivity. Specifically, the microorganism is, for example, selected fromgenus represented by Achromobacter, Proteus, and Morganella.Particularly, Achromobacter sp. AJ2425, Proteus rettgeri IFO13501 andMorganella morganii IFO3168 are preferable as such microorganisms.

Herein, Achromobacter sp. AJ2425 has been deposited as follows.

Achromobacter sp. Strain AJ2425

(a) Accession No. FERM BP-8244 (transferred from FERM P-18786 to theInternational Patent Organism Depositary, Nov. 22, 2002).(b) Deposition date: Mar. 20, 2002(c) Depositary Organization: International Patent Organism Depositary,The Institute of Advanced Industrial Science and Technology (No. 6,Chuo, 1-1-1, Higashi, Tsukuba, Ibaraki, Japan (zip code: 305-8566))

Additionally, microorganisms deposited at the Institute forFermentation, Osaka (IFO) may be supplied and available from theInstitute for Fermentation, (2-17-85, Tomimoto-cho, Yodogawa-ku, Osaka,Japan (zip code: 532-8686)).

These microorganisms may be microbial strains newly separated from thenatural resources, such as in soil or from plants or may be microbialstains artificially grown by treatment with mutagenic chemicals orrecombinant DNA technology.

The method for culturing the microorganism that produces the enzyme foruse in reaction 1 may be performed using general culture media for usein the field, culture media containing carbon sources, nitrogen sources,inorganic salts, trace metal salts, vitamins and the like.

As the carbon sources, for example, any carbon source that is compatiblewith the microorganism may be used. Exemplary carbon sources includesugars such as glucose, sucrose and dextrin, alcohols such as sorbitol,ethanol and glycerol, organic acids such as fumaric acid, citric acid,acetic acid and propionic acid and salts thereof, hydrocarbons such asparaffin or mixtures thereof described above.

As the nitrogen sources, for example, ammonium sulfate, ammoniumchloride, urea, yeast extract, meat extract, corn steep liquor andcasein-hydrolyzed products or mixtures thereof may be used.

As a specific culture medium composition, for example, a culture mediumcontaining 1.0% glucose, 0.3% ammonium sulfate, 1.0% powder yeastextract, 1.0% peptone, 0.1% KH₂PO₄, 0.3% K₂HPO₄, 0.05% MgSO₄.7H₂O,0.001% FeSO₄.7H₂O, and 0.001% MnSO₄.4H₂O (pH 7.0) may be used.

Additionally, microbial cells having a high ability to converttryptophan to indole-3-pyruvic acid may sometimes be obtained by addingL-amino acid or D-amino acid as an enzyme-inducing agent to the culturemedium.

Detergents such as Triton X and Tween and organic solvents such astoluene and xylene may be used to enhance the permeability of microbialcells for incorporation of the substrate.

As to the culture temperature, generally, the reaction is performedwithin a range where a microorganism may grow. In view thereof, thetemperature is generally in the range of about 20 to 45° C., preferably25 to 37° C.

As to the pH of the culture medium, the pH is preferably adjusted toabout 3 to 10, more preferably about 4 to 8.

As to the aeration condition, the condition should be set to a conditionsuitable for the growth of a microorganism to be used. Preferably,microorganism growth (and reaction) occurs under aerobic conditions.

As to the culture period, the reaction is generally conducted for about12 to 120 hours, preferably about 16 to 96 hours.

(A-2) Reaction Conditions for Reaction 1

Reaction 1 includes producing indole-3-pyruvic acid from tryptophan inthe presence of an enzyme catalyst and treating the resulting reactionsolution with any one method selected from deaeration treatment,deoxygen treatment and pH adjustment up to pH 2 at maximum to collectindole-3-pyruvic acid.

In reaction 1, the term “in the presence of an enzyme” means allowingthe enzyme to exist in the reaction system while the enzyme in an activestate to enable the conversion of tryptophan to indole-3-pyruvic acid.In other words, the enzyme may exist in the reaction system as long asthe enzyme is at any active state to enable converting tryptophan toindole-3-pyruvic acid. For example, the enzyme may be added solely tothe reaction system (i.e., an isolated or purified enzyme), or amicroorganism possessing the enzyme activity (e.g., microorganismgenerating the enzyme, cells transformed with recombinant DNA, etc.), aculture of the microorganism (e.g., liquid culture, solid culture,etc.), a culture medium (e.g. prepared by preliminarily eliminatingmicrobial cells from the culture) or a treated product of the culturemay be added to the reaction system. When a culture of a microorganismis used, reaction 1 may be progressed concurrently with culturing themicroorganism. Otherwise, reaction 1 may be performed using a culturefor obtaining the enzyme.

Herein, the “treatment” means a treatment for the purpose of recoveringenzymes in microbial cells. The treatment includes, for example,treatments with ultrasonication, glass beads, French press, andfreeze-drying and treatments with lytic enzymes, organic solvents,detergents or the like. Additionally, treated products after thesetreatments are further treated by routine methods (e.g., liquidchromatography, ammonium sulfate fractionation, etc.) to prepare a crudeenzyme fraction or a purified enzyme, which may be used satisfactorily,when the fraction or the enzyme has an ability to be required.

When using the culture or the treated product thereof, the culture orthe treated product may be included in carrageenan or polyacrylamide ormay be immobilized on a film of polyether sulfone or regeneratedcellulose, prior to use.

The microbial cell or a treated product of the microbial cell may beused at an amount (effective amount) sufficient to exert the intendedeffect in case of a given reaction. As to the effective amount, a personskilled in the art may readily determine the amount by a simplepreliminary experiment. For rinsed wet microbial cells, for example, theamount preferably ranges 1 to 40 g per 100 ml of reaction solution.

As the substrate tryptophan, any of the L form, the D form and the DLform may be used. From the ready availability and price, the L form ispreferred. Tryptophan is integrally, intermittently or continuouslyadded within a concentration range without suppression of the intendedreaction. As to the addition method, tryptophan may be directly added tothe microbial cells during culturing. Otherwise, the microbial cellsafter the culture are once separated, with which tryptophan is mixed.Tryptophan may be mixed with a treated product thereof. For addition,the substrate is added as an aqueous solution or slurry. For the purposeof increasing the solubility or promoting the dispersion, organicsolvents or detergents with no influence on the reaction may be mixed intryptophan prior to or concurrent with addition.

The reaction for use in accordance with the invention is performedwithin a pH range of preferably about pH 3 to 10, more preferably aboutpH 5 to 9 and within a temperature range of preferably 10 to 60° C.,more preferably about 20 to 40° C. for a reaction time of preferablyabout 0.5 to 120 hours, more preferably about 0.5 to 24 hours, whilestirring or while the reaction mixture is left to stand alone. Thesubstrate may be used at any concentration with no specific limitation,but may be preferably used at a concentration ranging from 0.1% to 10%.

As to the quantitative determination of tryptophan remaining in theliquid culture or in the reaction solution, the producedindole-3-pyruvic acid therein or the by-product indoleacetic acidtherein, these may be readily measured immediately using well-knownmethods by high-performance liquid chromatography.

The liquid culture accumulating the indole-3-pyruvic acid (reactionsolution) therein is treated by deaeration or deoxygen treatment tosuppress decomposition of indole-3-pyruvic acid. As a method fordeaeration treatment and deoxygen treatment, a process of substitutingthe gas (the whole or a part) contained in the reaction solution withinactive gases for example nitrogen and argon is listed.

Herein, the “deaeration treatment” means a procedure for eliminatingcomponents reactive with indole-3-pyruvic acid. These procedures includeremoving oxygen and hydrogen peroxide existing in the reaction solutionor lowering the concentrations thereof, a process for substituting thereaction solution with inactive gases or a process for putting thereaction solution to conditions under reduced pressure (i.e, usingaspirator and vacuum pump). Additionally, the “deoxygen treatment” meansa procedure for eliminating the dissolved oxygen in the reactionsolution or lowering the concentration thereof. Specifically, the methodof eliminating oxygen in the solution includes for example a process ofeliminating oxygen with inactive gas or a process of adding a deoxygenagent to the solution.

By substituting the reaction solution with inactive gas, the oxygenremaining in the reaction solution may be eliminated to terminate thereaction and additionally prevent the decomposition of the producedindole-3-pyruvic acid and the residual tryptophan. Herein, the “inactivegas” means a gas that does not directly or indirectly react withindole-3-pyruvic acid but effectively lowers the concentration and/oractivity of the components reactive with indole-3-pyruvic acid andtryptophan, such as oxygen or a trace amount of residual hydrogenperoxide. Examples of the inactive gas suitable for use in accordancewith the invention include nitrogen, argon and helium. The substitutionwith inactive gas may be performed immediately after the completion ofthe reaction. In case of the reaction using rinsed microbial cells, thesubstitution may be performed after the microbial cells are separated.

The method for charging inactive gas includes, for example, a process ofsubstituting the gaseous phase with inactive gas to lower the oxygenconcentration in the gaseous phase and a process of introducing inactivegas in the solution to eliminate the dissolved oxygen. The chargingmethod is not particularly limited. In regard to the oxygenconcentration in the gaseous phase, 5% or less, preferably 3% or less,more preferably 1% or less is adopted. Desirably, the oxygenconcentration in the solution is 1 ppm or less, preferably 0.1 ppm orless, more preferably 0.01 ppm or less.

Further, the reaction may be terminated and the decomposition ofindole-3-pyruvic acid may be suppressed by appropriately adding knownsubstances such as sodium sulfite having been known to have an effect onthe decrease of the dissolved oxygen concentration to the reactionsolution.

As the deoxygen agent in accordance with the present invention, sulfiteions may be used. As the source for sulfite ion, there may be used saltssuch as sodium sulfite, potassium sulfite, ammonium sulfite andsulfurous acid or hydrosulfite. These are preferably used at a sulfiteion or hydrosulfite concentration of preferably 20 ppm or more to 1% orless, more preferably 100 ppm or more to 0.5% or less.

The inactive gas substitution treatment and the method for addingdeoxygen agents to the solution may be performed in combination oreither one thereof may be done.

The indole-3-pyruvic acid produced by the reaction is collected from theliquid culture or the reaction solution by general methods, prior touse. For the collection thereof from the liquid culture or the reactionsolution, well-known procedures for general use in the field in suchcase, including filtration, centrifugation, vacuum concentration, ionexchange or adsorption chromatography or crystallization, may be used inappropriate combination, if necessary.

In one preferable embodiment of the invention, the pH of the reactionsolution is lowered, to thereby crystallize or precipitateindole-3-pyruvic acid. The resulting crystals may be directly separatedand collected from the mixture after the completion of the reaction.Concerning pH adjustment of the reaction solution, the pH is preferablyadjusted to 2 or less, more preferably adjusted to 1 or less.

In accordance with the invention, indole-3-pyruvic acid may be producedat a high production yield while the concentrations of the by-productketo acid and residual L-tryptophan are reduced in the solution. Bydirectly crystallizing indole-3-pyruvic acid under acidic conditions,therefore, the purification step may be simplified. In a more preferableembodiment of the invention, indole-3-pyruvic acid may be directlycrystallized by appropriately adding acids such as sulfuric acid andhydrochloric acid to the reaction solution. In accordance with theprocess of the present invention, indole-3-pyruvic acid may be producedat a high production yield while the concentrations of the keto acidby-product and residual tryptophan are reduced in the solution. Thus,the purification procedure may be simplified by directly crystallizingindole-3-pyruvic acid under acidic conditions.

For the adjustment to acidic conditions, the acid type to be used is notspecifically limited as long as the acid type serves to reduce the pH ofthe reaction solution to acidity. Examples of the acid to be usedinclude for example hydrochloric acid, sulfuric acid, nitric acid andphosphoric acid. A person skilled in the art may appropriately selectthe crystallization temperature, the amount of the acid to be used, thecrystallization period of time, and the method for adding the acid,within a range with no deterioration of the practice of the invention.

The crystallization temperature preferably ranges from −20° C. to 100°C., more preferably from 0° C. to 60+ C. may. As the amount of the acidto be used, an amount thereof to adjust the reaction solution topreferably pH 2 or less, more preferably pH 1 or less may be selected.The acid may be added and used so that the hydrogen ion concentration inthe solution after the acid addition is preferably about 0.01 to 10mol/L, more preferably about 0.1 to 1 mol/L.

As the crystallization period of time, preferably about 1 to 100 hours,more preferably about 1 to 24 hours may be selected.

[B] Reaction 2

Reaction 2 in accordance with the invention is a reaction forsynthesizing the precursor keto acid (IHOG) of monatin fromindole-3-pyruvic acid and pyruvic acid (or oxaloacetic acid). However,reaction 2 may also be used not only for the IHOG synthesis but also forthe synthesis of the substituted α-keto acid for use as the substrate inreaction 3 described below.

Specifically, reaction 2 may also be used widely as a reaction forproducing the substituted α-keto acid represented by formula (4)

from the substituted α-keto acid represented by formula (3)

and oxaloacetic acid or pyruvic acid.

The substituted α-keto acid of formula (4) as obtained via reaction 2may be used as the substrate for reaction 3 described below.

In formulae (3) and (4), R represents a substituent selected from thegroup consisting of an C2 to C8 alkyl group, a C1 to C8 alkoxyl group, aC2 to C9 carboxyalkyl group, an aryl group having up to 20 carbon atoms,an aralkyl group having up to 20 carbon atoms, a heterocyclicring-containing hydrocarbon group, and a hydroxyl group. When R includesan aromatic ring or a heterocyclic ring, the aromatic ring or theheterocyclic ring may be further substituted with halogen atoms (iodineatom, bromine atom, chlorine atom, fluorine atom, etc.), a hydroxylgroup, an alkyl group having up to 3 carbon atoms, an alkoxyl grouphaving up to 3 carbon atoms and an amino group.

R is preferably a phenylmethyl group or a 3-indolylmethyl group,particularly preferably a 3-indolylmethyl group. Specifically, thesubstituted α-keto acid of the general formula (3) is preferablyphenylpyruvic acid or indole-3-pyruvic acid, particularly preferablyindole-3-pyruvic acid. As the indole-3-pyruvic acid, theindole-3-pyruvic acid prepared by the process described in item [A] ofReaction 1 is preferable. However, reasonably, the process of preparingindole-3-pyruvic acid is not limited to this process.

In case that indole-3-pyruvic acid is used as the substituted α-ketoacid of the general formula (3), IHOG as the important intermediate formonatin production may be produced (reaction scheme (16)).

In case that phenylpyruvic acid is used as the substituted α-keto acidof the general formula (3), PHOG (4-phenylmethyl-4-hydroxy-2-oxoglutaricacid) as an intermediate keto acid for a monatin analog4-phenylmethyl-4-hydroxyglutamic acid (PHG) may be produced (scheme(17)).

The mode for carrying out reaction 2 is not specifically limited. Thisreaction may be either a chemical reaction system or an enzymaticsystem. The mode for carrying out reaction 2 is divided into chemicalreaction systems and enzymatic system, which are described in thefollowing order:

[B-1] chemical reaction system;

[B-2] enzymatic reaction system;

[I] enzymes for use in reaction 2;

(1) DNA encoding aldolase;

(2) properties of aldolase;

(3) Process of producing aldolase;

[II] reaction conditions for reaction 2.

(B-1) Chemical Reaction System

Reaction 2 using a chemical reaction system may be readily performed,using the method described below and the following Example 2, with nolimitation to the method.

For example, the substituted α-keto acid represented by formula (4) maybe produced by subjecting the substituted α-keto acid of formula (3) andoxaloacetic acid to a cross-aldol reaction and a decarboxylationreaction. The compound obtained by the aldol reaction is preliminarilyformed in the reaction system, which is an important intermediate.Without isolation of the compound, the following decarboxylation stepmay be progressed.

The conditions for the aldol reaction are readily determined with nodifficulty. The reaction may readily be progressed only when asubstituted pyruvic acid and oxaloacetic acid are allowed to interactwith each other in an appropriate solvent in the presence of aninorganic base or an organic base.

The type of the solvent to be used is not specifically limited, as longas the solvent is inactive to the reaction.

A person skilled in the art may appropriately select the reactiontemperature, the amount of base to be used, the reaction period of time,and the method for adding starting materials, within a range with nodeterioration of the practice of the invention.

The solvent preferably includes for example polar solvents such aswater, methanol, acetonitrile, and dimethylformamide.

If used, the base preferably includes hydroxides or carbonates ofinorganic bases (e.g., alkali metals or alkali earth metals, includinglithium hydroxide, sodium hydroxide, potassium hydroxide, sodiumcarbonate, potassium carbonate and calcium carbonate), and organic bases(e.g., triethylamine).

The reaction temperature preferably ranges from −20 to 100° C., morepreferably from 0 to 60° C.

For the reaction for decarboxylating the condensate from the aldolreaction, the reaction may be completed by spontaneous decarboxylation,but decarboxylation may be effectively performed by adding acid or metalion (or both) to the reaction solution. The acid for use in that caseincludes, for example, hydrochloric acid, sulfuric acid, phosphoricacid, acetic acid, p-toluenesulfonic acid, and solid acids such as ionexchange resins, while the metal ion includes, for example, transitionmetal ions such as nickel ion, copper ion, and iron ion. The reactiontemperature preferably ranges from −10 to 100° C., more preferably from0 to 60° C.

(B-2) Enzymatic Reaction System (I) Enzyme for Use in Reaction 2;

As the enzyme for use in reaction 2, any enzyme catalyzing the reactionfor synthesizing the substituted α-keto acid represented by formula (4)via the aldol condensation between the substituted α-keto acidrepresented by formula (3) and oxaloacetic acid or pyruvic acid may beused, with no specific limitation. In other words, any enzyme derivedfrom microorganisms or obtained by genetic recombination technology maybe satisfactory as long as the enzyme catalyzes the reaction.

The investigations of the inventors have verified that microbial strainsgenerating aldolase with the 4-phenylmethyl-4-hydroxy-2-oxoglutaric acid(PHOG)-decomposing activity exist in microorganisms belonging to thegenera Pseudomonas, Erwinia, Flavobacterium, and Xanthomonas.

The aldolase generated by these microorganisms catalyzes the reactionfor decomposing one PHOG molecule to generate one molecule ofphenylpyruvic acid and one molecule of pyruvic acid. The inventorsthought that the aldolase might possibly catalyze the reaction forsynthesizing 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid (IHOG)from indole-3-pyruvic acid and pyruvic acid (or oxaloacetic acid). Basedon this concept, the inventors isolated and purified aldolase fromcultured microbial cells of the microbial strains so as to identify thepresence of a novel aldolase. Additionally, the inventors found thatowing to the enzyme, IHOG was synthesized via the aldol condensationbetween indole-3-pyruvic acid and pyruvic acid (or oxaloacetic acid).

As microbial enzymes catalyzing the aldol condensation from twomolecules of α-keto acid (and substituted α-keto acid) as the substrate,two examples were reported in the related art. These reports used4-hydroxy-4-methyl-2-oxoglutarate aldolase from a bacterium of the genusPseudomonas and 4-hydroxy-2-oxoglutarate aldolase in E. coli, B.subtilis or the like. A report discloses that the former4-hydroxy-4-methyl-2-oxoglutarate aldolase catalyzes the reaction forgenerating 4-hydroxy-4-methyl-2-oxoglutarate (4-HMG) from two moleculesof pyruvic acid and the reaction for generating one molecule ofoxaloacetic acid and one molecule of pyruvic acid from4-oxalocitramalate (see Kiyofumi Maruyama, Journal of Biochemistry,1990, 108, pp. 327-333). Additionally, it is also known that the latter4-hydroxy-2-oxoglutarate aldolase catalyses the reaction for generating4-hydroxy-2-oxoglutarate (4HG) from one molecule of glyoxylic acid andone molecule of pyruvic acid.

However, heretofore, no report or no finding exists in regard to the4-phenylmethyl-4-hydroxy-2-oxoglutaric acid (referred to as PHOGhereinafter)-decomposing activity or about the synthetic activity of themonatin precursor keto acid (IHOG) from indole-3-pyruvic acid andpyruvic acid (or oxaloacetic acid) in any of these microbial strains. Itwas totally unknown whether or not the aldolase generated by thesemicrobial strains could be used for the synthetic route described above.

Prior to the findings of the inventors, in other words, no report existsregarding an example of synthetically preparing the precursor keto acid(IHOG) from indole-3-pyruvic acid and pyruvic acid (or oxaloaceticacid), using a microbial enzymatic system.

Additionally, the inventors purified the aldolase derived fromPseudomonas taetrolens ATCC4683 and determined the amino acid sequenceof the aldolase. Further, the inventors successfully achieved thesynthesis of a DNA molecule of about 30 bp as speculated from the aminoacid sequence of the aldolase, the isolation and recovery of a part ofthe DNA encoding the aldolase by PCR, and the isolation of thefull-length DNA encoding the aldolase derived from Pseudomonastaetrolens in the Pseudomonas taetrolens chromosomal gene libraries,using the resulting DNA fragment as probe.

SEQ ID No. 1 in the sequence listing shows the DNA encoding the aldolaseof the invention, as identified by the method described above.Additionally, SEQ ID Nos. 2 and 3 show the amino acid sequences of thealdolase encoded by the nucleotide sequence SEQ ID No. 1 in the sequencelisting. SEQ ID No. 2 in the sequence listing shows the amino acidsequence of the aldolase, which is encoded by the nucleotide sequence atthe 456-th to the 1118-th position in the nucleotide sequence SEQ ID No.1 in the sequence listing. Additionally, SEQ ID No. 3 in the sequencelisting shows the amino acid sequence of the aldolase, which is encodedby the nucleotide sequence at the 444-th to the 1118-th position in thenucleotide sequence SEQ ID No. 1. Any aldolase described as SEQ ID Nos.2 and 3 has the aldolase activity and catalyzes the reaction forsynthesizing 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid (IHOG)from one molecule of indole-3-pyruvic acid and one molecule of pyruvicacid (or oxaloacetic acid).

(1) DNA Encoding Aldolase

As described above, the aldolase gene of the nucleotide sequence SEQ IDNo. 1 in the sequence listing was isolated from the chromosomal DNA ofthe Pseudomonas taetrolens strain ATCC4683. The nucleotide sequence SEQID No. 1 in the sequence listing has 29% homology to the known4-hydroxy-4-methyl-2-oxoglutarate aldolase (name of the gene: proA)derived from the bacterium Pseudomonas ochraceae (see Maruyama K., etal., Biosci. Biotechnol. Biochem., 2001, 65 (12), pp. 2701-2709) interms of amino acid sequence. Herein, the homology is calculated, usinga gene analysis software “genetyx ver. 6” while various parameters wereemployed as they were initially set in the software package.

The method for obtaining the DNA encoding aldolase from analdolase-generating bacterium is now described.

First, the amino acid sequence of the purified aldolase is determined.Using the Edman method (Edman, P., Acta Chem. Scand., 1950, 4, p. 227),herein, the amino acid sequence may be determined. Alternatively, byusing a sequencer manufactured by Applied Biosystems Inc., the aminoacid sequence may also be determined. The aldolase derived fromPseudomonas taetrolens strain ATCC4683 of the invention was subjected tolimited proteolysis with protease. The resulting peptide fragments wereseparated and recovered by reverse-phase HPLC. The internal amino acidsequences of two of those fragments were determined, to consequentlyidentify the sequences SEQ ID Nos. 4 and 5.

Based on the amino acid sequences identified, the nucleotide sequence ofDNA encoding the sequences may be deduced. For determining thenucleotide sequence of DNA the universal genetic code was used.

Based on the deduced nucleotide sequence, DNA molecules of about 30 basepairs were synthetically prepared. The method for syntheticallypreparing the DNA molecule is disclosed in Tetrahedron Letters, 1981,22, p. 1859. Additionally, the DNA molecule may be syntheticallyprepared, using a synthesizer manufactured by Applied Biosystems Inc.The DNA molecule may be used as the probe for isolating the full-lengthDNA encoding the aldolase from the chromosomal gene libraries of thealdolase-generating microorganism. Otherwise, the DNA molecule may beused as the primer for amplifying the DNA encoding the aldolase of theinvention by PCR. Because the DNA amplified by PCR does not include thefull-length DNA encoding the aldolase, the DNA amplified by PCR is usedto isolate the full-length DNA encoding the aldolase from thechromosomal gene libraries of the aldolase-generating microorganism.

The PCR procedure is described by White, T. J., et al., Trends Genet. 5,1989, p. 185. The method for preparing chromosome DNA and the method forisolating an desired DNA molecule from a gene library using a DNAmolecule as probe are described in Molecular Cloning, 2nd edition, ColdSpring Harbor press, 1989.

The method for determining the nucleotide sequence of the isolated DNAencoding the aldolase is described in A Practical Guide to MolecularCloning, John Wiley & Sons, Inc., 1985. Further, the nucleotide sequencemay also be determined, using a DNA sequencer manufactured by AppliedBiosystems Inc. SEQ ID No. 1 in the sequence listing shows the DNAencoding the aldolase derived from the Pseudomonas taetrolens strainATCC4683.

It is to be understood that the DNA of SEQ ID No. 1 is not the only DNAencoding an aldolase catalyzing the reaction for the synthesis of IHOGfrom indole-3-pyruvic acid and pyruvic acid (or oxaloacetic acid) foruse in the present invention. Each species and each strain of the genusPseudomonas generating the aldolase catalyzing the reaction for thesynthesis of IHOG from indole-3-pyruvic acid and pyruvic acid (oroxaloacetic acid) should have difference in their nucleotide sequences,but nonetheless provide an aldolase that is embraced by the presentinvention.

Reasonably, even a DNA resulting from artificial mutation of thealdolase-encoding DNA isolated from the chromosome DNA of analdolase-generating bacterium may also be used for reaction 2, in casethat the artificial DNA encodes the aldolase. Site-specific mutationprocess described in Method. in Enzymol., 1987, p. 154 is frequentlyused as a process of adding such artificial mutation.

Additionally, a DNA hybridizing with a DNA of a nucleotide sequencecomplementary to the nucleotide sequence SEQ ID No. 1 in the sequencelisting under stringent conditions and encoding a protein with thealdolase activity may also be used for reaction 2. Herein, the term“stringent conditions” means conditions for forming so-called specifichybrid but excludes the formation of non-specific hybrids. Although itis difficult to clearly show the conditions in numerical figure, oneexample thereof is as follows: under the conditions, DNAs with highhomology of for example 50% or more, preferably 80% or more, morepreferably 90% or more and particularly preferably 95% or more mayhybridize to each other, but DNAs with lower homology cannot hybridizetogether. The term homology is preferably expressed as a valuecalculated while sequences for comparison are aligned so that the numberof the same bases may be the largest. Otherwise, the conditions areconditions for enabling hybridization at a salt concentrationcorresponding to the general rinse condition for Southern hybridization,namely 0.1×SSC, 0.1%×SDS at 37° C., preferably 0.1×SSC, 0.1%×SDS at 60°C., more preferably 0.1×SSC, 0.1%×SDS at 65° C.

Further, the term “aldolase activity” means any activity forsynthetically preparing IHOG from indole-3-pyruvic acid and pyruvic acid(or oxaloacetic acid). In case of a nucleotide sequence hybridizing witha nucleotide sequence complementary to the nucleotide sequence SEQ IDNo. 1 in the sequence listing under stringent conditions, the activityis at 10% or more, preferably 30% or more, more preferably 50% or more,still more preferably 70% or more of the aldolase activity of theprotein of the amino acid sequence SEQ ID No. 2 or 3 in the sequencelisting under conditions at 33° C. and pH 9.

Further, DNA encoding essentially the same protein as the aldolaseencoded by the DNA described as SEQ ID No. 1 may also be used forreaction 2. In other words, the following DNAs are also included in theDNA of the invention.

-   -   (a) DNA encoding the protein of the amino acid sequence SEQ ID        No. 2 in the sequence listing.    -   (b) DNA encoding the protein of an amino acid sequence prepared        after substitution, deletion, insertion, addition or inversion        of one or several amino acid residues in the amino acid sequence        SEQ ID No. 2 in the sequence listing and with the aldolase        activity.    -   (c) DNA encoding the protein of the amino acid sequence SEQ ID        No. 3 in the sequence listing.    -   (d) DNA encoding the protein of an amino acid sequence prepared        after substitution, deletion, insertion, addition or inversion        of one or several amino acid residues in the amino acid sequence        SEQ ID No. 3 in the sequence listing and with the aldolase        activity.

Herein, the term “one or several” means a range of amino acid residuesinvolving no severe deterioration of the steric configuration of theresulting protein or the aldolase activity, specifically including oneto 50, preferably one to 30, more preferably one to 10. As describedabove, additionally, the term “aldolase activity” means the activity forthe synthesis of IHOG from indole-3-pyruvic acid and pyruvic acid (oroxaloacetic acid). In case of an amino acid sequence prepared aftersubstitution, deletion, insertion, addition or inversion of one orseveral amino acid residues in the amino acid sequence SEQ ID No. 2 inthe sequence listing, the resulting aldolase activity under conditionsat 33° C. and pH 9 is at 10% or more, preferably 30% or more, morepreferably 50% or more, still more preferably 70% or more of thealdolase activity of the protein of the amino acid sequence SEQ ID No. 2or 3 in the sequence listing, under conditions at 33° C. and pH 9.

(2) Aldolase Properties

Then, the properties of the aldolase purified from the Pseudomonastaetrolens strain ATCC4683 is now described.

The aldolase derived from the Pseudomonas taetrolens strain ATCC4683 hasthe amino acid sequence SEQ ID No. 2 or 3 as clearly shown by theisolation and analysis of the gene described above. Reasonably, however,a protein of an amino acid sequence prepared after substitution,deletion, insertion, addition or inversion of one or several amino acidresidues in the amino acid sequence SEQ ID No. 2 or 3 in the sequencelisting and with the aldolase activity may also be used for reaction 2.

In other words, the following proteins (a) to (d) may also be used asthe enzyme catalyzing reaction 2.

-   -   (a) Protein of an amino acid sequence SEQ ID No. 2 in the        sequence listing.    -   (b) Protein of an amino acid sequence prepared after        substitution, deletion, insertion, addition or inversion of one        or several amino acid residues in the amino acid sequence SEQ ID        No. 2 in the sequence listing and with the aldolase activity.    -   (c) Protein of the amino acid sequence SEQ ID No. 3 in the        sequence listing.    -   (d) Protein of an amino acid sequence prepared after        substitution, deletion, insertion, addition or inversion of one        or several amino acid residues in the amino acid sequence SEQ ID        No. 3 in the sequence listing and with the aldolase activity.

Herein, the definitions of “several” and “aldolase activity” are thesame as described in the description in the item DNA encoding aldolase(1).

Such aldolase may catalyze the reaction for synthetically preparing4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid (IHOG) via the aldolcondensation from indole-3-pyruvic acid and pyruvic acid (or oxaloaceticacid).

The aldolase activity of the aldolase may be assayed by measuring theamount of IHOG generated from indole-3-pyruvic acid and pyruvic acid (oroxaloacetic acid) by high performance liquid chromatography (HPLC).

Specifically, the aldolase activity may be estimated by adding aldolaseto a reaction solution of 100 mM buffer, 50 mM indole-3-pyruvic acid,250 mM pyruvic acid, 1 mM MgCl₂, and 1 v/v % toluene, for reaction undershaking at 33° C. for 4 hours and measuring the amount of IHOG generatedby HPLC.

IHOG may be determined by HPLC using “Inertsil ODS-2” (5 μm, 4.6×250 mm)manufactured by GL Sciences, Inc. One example of the analyticalconditions is shown below.

Mobile phase: 40 v/v % acetonitrile/5 mM phosphate dihydrogentetrabutylammonium solutionFlow rate: 1 ml/minColumn temperature: 40° C.

Detection: UV 210 nm.

The enzymatic and chemical properties of the aldolase from Pseudomonastaetrolens as measured by the aforementioned analytical method, which isfurther described below.

The aldolase derived from Pseudomonas taetrolens may catalyze thereaction for synthetically preparing IHOG via the aldol condensation ofindole-3-pyruvic acid and pyruvic acid (or oxaloacetic acid). Asmicrobial enzymes catalyzing the aldol condensation from two moleculesof α-keto acid (or substituted α-keto acid) as the substrate, so far,two such enzymes are reported, which are4-hydroxy-4-methyl-2-oxoglutarate aldolase from genus Pseudomonas and4-hydroxy-2-oxoglutarate aldolase existing in E. coli and B. subtilis.However, absolutely no finding or report exists about the formerconcerning the interaction thereof with PHOG or IHOG. Therefore, todate, it has been unknown whether or not PHOG (and IHOG) may besynthetically prepared, using the enzyme. Additionally, noPHOG-decomposing activity was observed in the latter, and therefore, thePHOG (and IHOG) synthesis using the enzyme was impossible. In otherwords, the aldolase derived from Pseudomonas taetrolenscharacteristically catalyzes the reaction for the synthesis of IHOG viathe aldol condensation of indole-3-pyruvic acid and pyruvic acid (oroxaloacetic acid), unlike the aldolase reported so far.

The optimum pH of the aldolase derived from Pseudomonas taetrolens isaround 9 at 33° C.

The molecular weight of the aldolase derived from Pseudomonas taetrolensas measured by gel filtration was about 146 kDa and was about 25 kDameasured by SDS-PAGE. Therefore, the aldolase of the invention willpossibly form a homohexamer composed of a subunit with a molecularweight of about 25 kDa.

(3) Process of Preparing Aldolase

The process of preparing the aldolase is now described below. Theprocess of preparing the aldolase for use in reaction 2 in accordancewith the invention includes two processes, namely (i) a process ofgenerating and accumulating the aldolase by microbiologically culturingan aldolase-generating bacterium and (ii) a process of generating andaccumulating the aldolase by forming a transformant generating thealdolase by recombinant DNA technology and culturing the transformant.

(i) The Process of Generating and Accumulating the Aldolase by MicrobialCulturing

For the process of generating and accumulating the aldolase bymicrobiologically culturing an aldolase-generating bacterium, themicroorganism as a source for recovering the aldolase includes forexample microorganisms belong to the genera Pseudomonas, Erwinia,Flavobacterium, and Xanthomonas.

Among the genera Pseudomonas, Erwinia, Flavobacterium, and Xanthomonas,any microorganism generating aldolase catalyzing the reaction for thesynthesis of the precursor keto acid (IHOG) from indole-3-pyruvic acidand pyruvic acid (or oxaloacetic acid) may be used in accordance withthe invention. Preferably, the microorganism is selected fromPseudomonas taetrolens ATCC4683, Pseudomonas coronafaciens AJ2791,Pseudomonas desmolytica AJ1582, Erwinia sp. AJ2917, Xanthomonas citriAJ2797, and Flavobacterium rhenanum AJ2468. Particularly among them,Pseudomonas taetrolens ATCC4683 and Pseudomonas coronafaciens AJ2791 arepreferable. The depositary organizations of these microorganisms areshown below.

(1) Pseudomonas coronafaciens strain AJ2791

-   -   (a) Accession No. FERM BP-8246 (transferred from FERM P-18881 to        the International Depositary, Nov. 22, 2002)    -   (b) Deposition Date: Jun. 10, 2002    -   (c) Depositary: International Patent Organism Depositary, The        Institute of Advanced Industrial Science and Technology (No. 6,        Chuo, 1-1-1, Higashi, Tsukuba, Ibaraki, Japan)

(2) Pseudomonas desmolytica AJ1582

-   -   (a) Accession No. FERM BP-8247 (transferred from FERM P-18882 to        the International Depositary, Nov. 22, 2002)    -   (b) Deposition Date: Jun. 10, 2002    -   (c) Depositary: International Patent Organism Depositary, The        Institute of Advanced Industrial Science and Technology (No. 6,        Chuo, 1-1-1, Higashi, Tsukuba, Ibaraki, Japan)

(3) Erwinia sp. AJ2917

-   -   (a) Accession No. FERM BP-8245 (transferred from FERM P-18880 to        the International Depositary, Nov. 22, 2002)    -   (b) Deposition Date: Jun. 10, 2002    -   (c) Depositary: International Patent Organism Depositary, The        Institute of Advanced Industrial Science and Technology (No. 6,        Chuo, 1-1-1, Higashi, Tsukuba, Ibaraki, Japan)

(4) Flavobacterium rhenanum AJ2468

-   -   (a) Accession No. FERM BP-1862    -   (b) Deposition Date: Sep. 30, 1985    -   (c) Depositary: International Patent Organism Depositary, The        Institute of Advanced Industrial Science and Technology (No. 6,        Chuo, 1-1-1, Higashi, Tsukuba, Ibaraki, Japan)

(5) Xanthomonas citri AJ2797

-   -   (a) Accession No. FERM BP-8250 (transferred from FERM P-4347 to        the International Depositary, Nov. 27, 2002)    -   (b) Deposition Date: Sep. 30, 1985    -   (c) Depositary: International Patent Organism Depositary, The        Institute of Advanced Industrial Science and Technology (No. 6,        Chuo, 1-1-1, Higashi, Tsukuba, Ibaraki, Japan)

The mode for culturing the microorganism as a source for recovering thealdolase may be any of liquid culture and solid culture. An industriallyadvantageous mode is submerged aeration culture. As the nutrient sourcein nutritious culture media, those generally used for microbial culture,such as carbon sources, nitrogen sources, inorganic salts and othertrace nutrient sources may be used. Any nutrient sources compatible withthe microbial strains may be used.

For the aeration condition, aerobic conditions are adopted. The culturetemperature may be within a range for microbial growth to generate thealdolase. Thus, no strict conditions exist therefor. Generally, thetemperature is 10 to 50° C., preferably 30 to 40° C. The culture timeperiod varies, depending on other culture conditions. For example, theculture time is up to a period involving the maximum aldolasegeneration, which is for example 5 hours to 7 days, preferably about 10hours to about 3 days.

After the culture, the microbial cells are harvested by centrifugation(for example, 10,000×g, 10 minutes). Because aldolase mostly exists inmicrobial cells, the microbial cells are preferably disrupted or lysed,for aldolase solubilization. For the microbial disruption, treatmentssuch as ultrasonic disruption, French press disruption, and glass beaddisruption may be used. In case of lysis, additionally, a treatment withegg white lysozyme or peptidase or an appropriate combination of suchtreatments may be used.

For purifying the aldolase derived from the aldolase-generatingbacterium, the enzyme-solubilized solution is used as a startingmaterial for the purification. When undisrupted or non-lysed residue maybe left, the solubilized solution is again treated by centrifugationprocedures to remove the precipitating residue, which is ratheradvantageous for the purification.

For purifying the aldolase, any routine process generally used forenzyme purification may be used, including for example ammonium sulfatesalting-out process, gel filtration chromatography, ion exchangechromatography, hydrophobic chromatography and hydroxyapatitechromatography. Consequently, aldolase-containing fractions with ahigher activity may be obtained.

(ii) Process by Recombinant DNA Technology

The process of preparing the aldolase by recombinant DNA technology isnow described. Numerous examples of preparing useful proteins such asenzymes and physiologically active substances using recombinant DNAtechnology are known in the art. Using recombinant DNA technology,useful proteins naturally occurring at trace amounts may be prepared ata mass scale.

Any DNA to be conjugated to a vector DNA may be satisfactory if the DNAmay express the aldolase.

As an example of the aldolase gene conjugated to a vector DNA, herein,(1) the DNA described in the item “DNA encoding aldolase” may be used.

In case of mass protein preparation using recombinant DNA technology,preferably, the protein is associated together in a transformant to forma protein inclusion body. The advantages of the expression andpreparation process are as follows. The intended protein may beprotected from digestion with protease in microbial cells and theintended protein may be purified in a simple manner by centrifugationprocedures following microbial cells disruption.

The protein inclusion body obtained in such manner is solubilized with aprotein-denaturing agent and is treated by a procedure for regeneratingthe activity by removing the denaturing agent, and restoring the proteinto a physiologically active (correctly folded) state. For example, thereare many examples such as regeneration of human interleukin-2 activity(JP-A-61-257931).

So as to obtain the active type protein from such protein inclusionbody, a series of procedures such as solubilization and activityregeneration are needed and are more complicated than those for directlygenerating the active type protein. In case of mass preparation of aprotein with influences on the growth of a microbial cell in themicrobial cell, the influences may be suppressed by accumulating theprotein in the form of an inactive protein inclusion body in themicrobial cell.

The process of mass preparation of an intended protein in the form of aninclusion body includes single expression of the intended protein undercontrols of a strong promoter, and a process of expressing the intendedprotein in the form of a fusion protein with a protein known to beexpressed at a large scale.

After the expression thereof as a fusion protein, so as to remove thefusion protein and obtain the intended protein effectively, recognitionsequences of restriction proteases should be arranged at appropriatesites.

In case of mass protein preparation using recombinant DNA technology,for example, microbial cells, actinomycetes cells, yeast cells, funguscells, plant cells and animal cells may be used as host cells fortransformation. The microbial cells for which host-vector systems arenow developed include for example microorganism of genus Escherichia,microorganism of genus Pseudomonas, microorganism of genusCorynebacterium, and microorganism of Bacillus. Preferably, Escherichiacoli is used because significant knowledge exists regarding how to useEscherichia coli for protein generation at a large scale. A process ofpreparing aldolase using transformed Escherichia coli is now described.

As the promoter for expressing the DNA encoding the aldolase, generally,promoters for Escherichia coli for exogenous protein preparation may beused and include for example strong promoters such as T7 promoter, trppromoter, lac promoter, tac promoter and PL promoter.

For generation of the aldolase in an inclusion body of the fusionprotein, a gene encoding another protein, preferably a hydrophilicpeptide is conjugated to the upstream or downstream of the aldolasegene, to prepare a fusion protein gene. The gene encoding such anotherprotein may satisfactorily be any gene capable of increasing theaccumulation of the fusion protein and raising the solubility of thefusion protein after denaturation and regeneration steps. For example,T7 gene 10, β-galactosidase gene, the gene of the dehydrofolate-reducingenzyme, interferon γ gene, interleukin-2 gene and prochymosin gene arecandidates thereof.

For conjugation of genes encoding the “other” protein to the geneencoding the aldolase, the other genes should have the same readingframe for their codons. These genes are conjugated at appropriaterestriction sites or are conjugated, using synthetic DNA of anappropriate sequence.

For increasing the generated amount, further, a terminator as atranscription termination sequence is preferably conjugated to thedownstream of the fusion protein gene. The terminator includes forexample T7 terminator, fd phage terminator, T4 terminator, theterminator for tetracycline resistant gene, and the terminator forEscherichia coli trpA gene.

The vector for introducing the gene encoding the aldolase or a geneencoding a fusion protein of the aldolase with another protein ispreferably a multi-copy type plamid and includes for example a plasmidwith an origin of replication as derived from Col E1, such as pUC-baseplasmid, pBR322-base plasmid or derivatives thereof. Herein, the term“derivative” in the context of plasmids means such plasmids denatured bynucleotide substitution, deletion, insertion, addition or inversion.Herein, the term denaturation means mutagenic treatment with mutagensand UV irradiation or spontaneous mutation.

For transformant screening, further, the vector preferably has markerssuch as ampicillin resistant gene. As such plasmid, expression vectorswith strong promoters are commercially available (pUC series(manufactured by TAKARA BIO INC.), pPROK series (manufactured byClontech Laboratories, Inc.), pKK233-2 (manufactured by ClontechLaboratories, Inc.), etc.).

A DNA fragment, in which a promoter, the gene encoding the aldolase or agene encoding a fusion protein of the aldolase with another protein anda terminator are sequentially conjugated together in this order, isconjugated to a vector DNA to form a recombinant DNA.

Using the recombinant DNA, Escherichia coli is transformed. Theresulting Escherichia coli is cultured to express and generate thealdolase or the fusion protein of the aldolase with another protein. Asthe host to be transformed, strains generally used for the expression ofexogenous gene may be used. Particularly, the Escherichia coli stainsJM109 (DE3) and JM109 are preferable. The process for suchtransformation and the method for screening the resulting transformantare described in Molecular Cloning, 2-nd edition, Cold Spring Harborpress, 1989 and the like.

In case of the expression in the form of fusion protein, the aldolasemay satisfactorily be excised using restriction proteases such as bloodcoagulation Factor Xa and kallikrein those recognizing sequences neverexisting in aldolase as the recognition sequences.

As the generation culture medium, culture media for general use inculturing Escherichia coli, such as M9-casamino acid culture medium andLB culture medium may be used. Additionally, the culture conditions andthe conditions for inducing the generation are appropriately selected,depending on the types of the marker, promoter, and host bacterium forthe vector used.

For recovering the aldolase or the fusion protein of the aldolase withanother protein, the following processes are used.

When the aldolase or the fusion protein is solubilized in microbialcells, the microbial cells are once recovered, which are then disruptedor lysed for use as a crude enzyme solution. If necessary, further, thealdolase and the fusion protein thereof may be purified by generalmethods such as precipitation, filtration and column chromatography,prior to use. In this case, purification methods using antibodiesagainst the aldolase or the fusion protein may also be used.

A protein inclusion body, when formed, is solubilized with a denaturingagent. The protein inclusion body may be solubilized together with themicrobial cell protein. In terms of the following purificationprocedure, however, the inclusion body is preferably taken out and thensolubilized. The inclusion body may be recovered from the microbialcells by known methods in the related art. For example, the microbialcells are disrupted to recover the inclusion body by centrifugationprocedures. The denaturing agent for solubilizing the protein inclusionbody includes for example guanidine hydrochloride (for example, 6M, pH 5to 8) and urea (for example, 8 M).

By removing these denaturing agents by incremental dialysis and thelike, the resulting protein may be regenerated as a protein with theactivity. As the dialysis solution for use in the dialysis, Tris-HClbuffer and phosphate buffer may be used. The concentration includes forexample 20 mM to 0.5 M at pH 5 to 8.

The protein concentration at the regeneration step is preferablysuppressed to about 500 μg/ml or less. So as to suppress theself-crosslinking of the regenerated aldolase, the temperature for thedialysis is preferably 5° C. or less. The process of removing thedenaturing agents includes dilution process and ultrafiltration processother than the dialysis process. Using any of these processes, theregeneration of the activity may be expected.

In case that the aldolase gene is derived from microorganism of thegenus Pseudomonas, additionally, the aldolase may be expressed andgenerated in a host microorganism of genus Pseudomonas as a preferableembodiment. For example, Shi-En Lu, et al. reported a transformation andexpression method in Pseudomonas syringae as the host cell (FEMSMicrobiology Letters, 2002, 210, pp. 115-121). Additionally, Olsen, R.H., et al. reported about the transformation and expression method inPseudomonas aeruginosa (Journal of Bacteriology, 1982, 150, pp. 60-69).Further, Grapner, S., et al. report about the transformation andexpression method in Pseudomonas stutzeri (Biomol. Eng., 2000, 17, pp.11-16). However, microorganisms of genus Pseudomonas as host cells forthe expression of the aldolase are not limited to those recited herein.

Concerning the vector for introducing the aldolase gene intomicroorganism of genus Pseudomonas, a plasmid with an replication onfunctioning inside cells of microorganism of the genus Pseudomonas maybe used. For example, Eza Kalyaeva, et al. report the plasmid pKLH4.05with the replicon TFK functioning in Pseudomonas aeruginosa.Additionally, so-called vectors for wide host ranges may also be used,which is used for transformation of Gram-negative microorganism. It isknown that among these vectors, for example, RK404 (Ditta, G., et al.,Plasmid, 1985, 13, pp. 149-153) and RSF1010 (Frey, J., et al., Gene,1982, 24, pp. 289-296) function in Pseudomonas microorganism.

When the DNA of SEQ ID No. 1 in the sequence listing is used as the DNAencoding the aldolase, the aldolase of the amino acid sequence SEQ IDNo. 2 or 3 is generated.

(II) Reaction Conditions for Reaction 2

The reaction conditions for reaction 2 when using an enzymatic systemare described below.

As the enzyme catalyzing reaction 2, any enzyme catalyzing the reactionfor synthesizing the substituted α-keto acid represented by formula (4)via the aldol condensation of the substituted α-keto acid represented byformula (3) and oxaloacetic acid or pyruvic acid may be used, with nospecific limitation. In other words, any enzyme derived frommicroorganisms or obtained by genetic engineering technology may besatisfactory, as long as the enzyme catalyzes the reaction.

As such enzyme, the aldolase described in the item (I) Enzyme for use inreaction 2 is preferable. In this case, the aldolase obtained byculturing microbial cells generating the aldolase catalyzing reaction 2from genera Pseudomonas, Erwinia, Flavobacterium and Xanthomonas may beused. Alternatively, the aldolase obtained by preparing a transformantgenerating aldolase catalyzing the reaction by recombinant DNAtechnology and then culturing the transformant may be used.

The term “in the presence of an enzyme” in reaction 2 means that anenzyme is allowed to exist in the reaction system, while the enzyme isat its state to enable catalyzing the reaction for syntheticallypreparing the substituted α-keto acid represented by formula (4) fromthe substituted α-keto acid represented by formula (3) and oxaloaceticacid or pyruvic acid. For example, the enzyme may singly be added to thereaction system or a microorganism with the activity of the enzyme(e.g., aldolase-generating microorganism, cells transformed byrecombinant DNA, etc.), a culture of the microorganism (e.g., liquidculture, solid culture, etc.), a culture media (e.g., the culture fromwhich microbial cells are removed), and treated products of the culturemay be added to the reaction system.

When the culture of the microorganism is used, reaction 2 is progressedwhile the microorganism is simultaneously cultured. Otherwise, reaction2 is performed using the culture prepared by preliminary culturing so asto obtain the enzyme. Additionally, the term “treatment” means treatmentfor the purpose of recovering the enzyme from microbial cells andincludes for example treatments with ultrasonication, glass bead, Frenchpress, and freeze-drying and treatments with lysed enzymes, organicsolvents, detergents or the like. Additionally, any crude fractionatedenzyme or purified enzyme as prepared by further processing the treatedproduct after these treatment by routine methods (e.g., liquidchromatography, ammonium sulfate fractionation, etc.) may besatisfactory, as long as the crude or purified enzyme has the abilityrequired.

When the substituted α-keto acid of formula (4) is produced using thealdolase-generating bacterium or a cell transformed by recombinant DNA,the substrates may be added to the liquid culture during the culture.Otherwise, any of the microbial cells separated from the liquid cultureand the rinsed microbial cells may be used. Additionally, the microbialcell-treated product prepared by disrupting or lysing the microbialcells may be used as it is or the aldolase recovered from the microbialcell-treated product is used as a crude enzyme solution, from which theenzyme is purified and used as well.

When using the culture or the treated product, further, the culture orthe treated product may be included in carrageenan and polyacrylamide ormay be immobilized on a film of polyether sulfone and regeneratedcellulose for use.

For progressing reaction 2 in the presence of an enzyme, a reactionsolution containing at least one of the substituted α-keto acidrepresented by formula (3), oxaloacetic acid or pyruvic acid and theenzyme catalyzing reaction 2 is adjusted to an appropriate temperatureof 20 to 50° C., which is then left to stand alone or is shaken oragitated while the pH is kept at 6 to 12 for 30 minutes to 5 days.

The reaction velocity may be increased by adding divalent cations suchas Mg²⁺, Mn²⁺, Ni²⁺, and Co²⁺ to the reaction solution. From thestandpoint of cost, preferably, Mg²⁺ may sometimes be used.

For adding these divalent cations to the reaction solution, any saltthereof may be used as long as the salt never inhibits the reaction.Preferably, MgCl₂, MgSO₄, MnSO₄ or the like may be used. A personskilled in the art may determine the concentration of these divalentcations to be added, by simple preliminary experiments. The divalentcations may be added within a range of 0.01 mM to 10 mM, preferably 0.1mM to 5 mM, more preferably 0.5 mM to 2 mM.

One example of the reaction conditions preferable for performingreaction 2 is described below. To the reaction solution consisting of100 mM buffer, 50 mM indole-3-pyruvic acid, 250 mM pyruvic acid, 1 mMMgCl₂, and 1 v/v % toluene, rinsed E. coli microbial cells expressingthe aldolase are added as the enzyme source to 10 w/v %, for reactionunder shaking at 33° C. for 4 hours, to obtain4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid (IHOG).

The resulting substituted α-keto acid of formula (4) may be separatedand purified by known methods. For example, the known methods include amethod of contacting the substituted α-keto acid with an ion exchangeresin to adsorb basic amino acids, and eluting and subsequentlycrystallizing the resulting substituted α-keto acid; and a method ofeluting the substituted α-keto acid, decoloring and filtering thesubstituted α-keto acid with active charcoal and then crystallizing thesubstituted α-keto acid.

Via reaction 2, the precursor keto-acid (IHOG) useful as an intermediatefor monatin synthesis may be generated from indole-3-pyruvic acid andpyruvic acid (or oxaloacetic acid).

[C] Reaction 3

The reaction 3 of the invention is a reaction related to monatinproduction and is preferably used for the synthesis of monatin from theprecursor keto acid (IHOG). However, the reaction 3 may be used not onlyfor the synthesis of monatin but also may be used widely for thereaction of producing glutamate derivatives of formula (2) from thesubstituted α-keto acid of formula (1).

Herein, R¹ and R² independently represent a substituent selected fromthe group consisting of a hydrogen atom, a C1 to C8 alkyl group, a C1 toC8 alkoxyl group, a C2 to C9 carboxyalkyl group, an aryl group having upto 20 carbon atoms, an aralkyl group having up to 20 carbon atoms, aheterocyclic group-containing hydrocarbon group, and a hydroxyl group.When either of R¹ and R² represents a hydrogen atom, the other is not ahydrogen atom, a methyl group or an ethyl group. When either one of R¹and R² represents a hydroxyl group, the other is not a hydrogen atom ora methyl group.

The aromatic ring or the heterocyclic ring contained in the substituentR¹ in the formula may contain at least one of halogen atoms, a hydroxylgroup, alkyl groups with up to 3 carbon atoms, alkoxyl group with up to3 carbon atoms, and an amino group.

Among the foregoing, preferably, R¹ is selected from the groupconsisting of a C₂ to C₄ alkyl group, a C₂ to C₄ carboxyalkyl group, aphenylmethyl group and a 3-indolylmethyl group (the benzene ring or theindole ring may additionally contain at least one of halogen atoms(iodine atom, bromine atom, chlorine atom, fluorine atom, etc.)), ahydroxyl group, an alkyl group having up to 3 carbon atoms, an alkoxylgroup having up to 3 carbon atoms and an amino group) and R² is ahydroxyl group. More preferably, R¹ is a phenylmethyl group or a3-indolylmethyl group and R² is a hydroxyl group.

In case that R¹ is a 3-indolylmethyl group and R² is a hydroxyl group,i.e. that IHOG (4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid) isused as the substituted α-keto acid of formula (1), monatin may beobtained as the glutamate derivative of formula (2).

In case that R¹ is a phenylmethyl group and R² is a hydroxyl group, i.e.that PHOG (4-phenylmethyl-4-hydroxy-2-oxoglutaric acid) is used as thesubstituted α-keto acid of ormula (1), a monatin analog4-phenylmethyl-4-hydroxy-glutamic acid (PHG) may be obtained as theglutamate derivative of formula (2).

As the substituted α-keto acid represented by formula (1) as thesubstrate, the substituted α-keto acid of formula (4) as obtained by theprocess described in the item [B] of Reaction 2 is preferably used. Morepreferably, IHOG prepared by the process in the item [B] of Reaction 2,using indole-3-pyruvic acid prepared by the process described in theitem [A] of Reaction 1 is used. However, reasonably, the process ofpreparing the substituted α-keto acid represented by formula (1) is notlimited to these processes described above.

Reaction 3 utilizes the reaction of the enzyme catalyzing the reactionfor generating an amino acid corresponding to the substituted α-ketoacid as the substrate. Reaction 3 relates, for example, to a process ofproducing glutamate derivatives by reacting a protein catalyzingtransamination or a microorganism generating the protein. Herein, theterm “transamination” means a reaction for converting a precursor ketonecompound to the corresponding amino compound by transferring the aminogroup of the donor substrate to the ketone group of the acceptorcompound.

The mode for carrying out the reaction 3 of the invention is nowdescribed in detail in the following order.

(C-1) Enzyme for use in reaction 3

(C-2) Reaction conditions for reaction 3.

(C-1) Enzyme catalyzing the reaction for generating amino acid

For reaction 3, the enzyme catalyzing the reaction for generating anamino acid corresponding to the substituted α-keto acid as the substrateindividually includes for example a transaminase as an enzyme catalyzingtransamination and additionally includes a dehydrogenase as the enzymecatalyzing the reductive amination of the keto acid. The transaminasefor use in reaction 3 is satisfactorily an enzyme catalyzing thereaction for generating a glutamate derivative from a correspondingsubstituted α-keto acid and an amino donor as the starting materials.Via the action of such enzyme, the substituted α-keto acid representedby formula (1) may be converted to the corresponding glutamatederivative (represented by formula (2)).

As the amino donor, then, a compound containing an amino group is used.For example, the compound includes amino compounds such as L-amino acidsand D-amino acids naturally occurring and non-naturally occurring.Specifically, the amino acids include for example glutamic acid,aspartic acid, alanine, tryptophan, phenylalanine, isoleucine, leucine,tyrosine, valine, arginine, asparagine, glutamine, methionine,ornithine, serine, cysteine, histidine and lysine. The amino donor to beadded for the reaction may be a single one type or a mixture of pluraltypes of such donors.

Generally, L-amino acid transaminase generates an intended L-amino acidby transferring the amino group of an L-amino acid donor to theprecursor keto acid, while D-amino acid transaminase generates anintended D-amino acid by transferring the amino group of a D-amino aciddonor to the precursor keto acid. Via the selection of such enzyme, anoptical isomer of a glutamate derivative to be generated may also beselected. For example, the reaction with D-amino acid transaminase inthe presence of D-amino acids such as D-alanine, D-glutamic acid, andD-aspartic acid may selectively generate D-glutamate derivatives fromthe precursor keto acid.

As described above, monatin as one of glutamate derivatives as theobject of the invention includes three optical isomers in addition tothe naturally occurring type (2S,4S). It has been confirmed that any ofthese isomers has a sweetness intensities several hundreds-fold to somethousands-fold that of sucrose. In one of the preferable embodiments ofthe present invention, the reaction of D-amino acid transaminase withthe precursor keto acid for monatin may generate the 2R form of monatinstereo-selectively, while the reaction of L-amino acid transaminase withthe precursor may generate the 2S form of monatin stereo-selectively. Inone of more preferable embodiments, the use of D-amino acid transaminasemay generate selectively the 2R form thereof as an isomer having highersweetness level.

When it is intended to use D-amino acid as an amino donor, herein, thecorresponding L-amino acid is added to the reaction solution, to allowthe amino acid to exist concurrently with an enzyme catalyzing theracemization reaction of the amino acid, so that the donor may besupplied as a D-amino acid donor. As such racemization enzyme,preferable examples thereof include alanine racemase, glutamic acidracemase, aspartic acid racemase, and phenylalanine racemase. In thiscase, L-alanine, L-glutamic acid, L-phenylalanine, L-aspartic acid orracemic mixtures of these L-amino acids may be added to the reactionsolution, while D-glutamate derivatives are under way of generation.

The enzyme catalyzing transamination may also be prepared by cultivatinga microorganism generating such enzyme. Such microorganism includes forexample microorganisms belonging to the genera Aeromonas, Agrobacterium,Alcaligenes, Bacillus, Beijerinckia, Escherichia, Proteus, Morganellaand Paenibacillus.

Specifically, these microorganisms include for example those describedbelow. In other words, the microorganism generating L-amino acidtransaminase with an activity for generating glutamate derivativesdescribed in formula (2) from the substituted α-keto acid described informula (1) include the following examples.

Aeromonas hydrophila IFO3820

Agrobacterium tumefaciens IFO3058

Alcaligenes faecalis ATCC8750

Beijerinckia indica ATCC9037

Escherichia coli ATCC12814

Proteus rettgeri IFO13501

Morganella morganii IFO3848

Additionally, the microorganisms generating D-amino acid transaminaseinclude the following examples.

Bacillus sphaericus ATCC10208

Bacillus pulvifaciens AJI1327

Paenibacillus larvae subsp. pulvifaciens ATCC13537

Bacillus macerans AJ1617

Paenibacillus macerans ATCC8244

Bacillus lentus AJ12699

Bacillus lentus ATCC10840

Herein, Bacillus macerans AJ1617 has been deposited as follows.

Bacillus macerans Strain AJ1617

-   -   (a) Accession No. FERM BP-8243 (transferred from FERM P-18653 to        the International Patent Organism Depositary, Nov. 22, 2002).    -   (b) Deposition date: Dec. 13, 2001    -   (c) Depositary Organization: International Patent Organism        Depositary, The Institute of Advanced Industrial Science and        Technology (No. 6, Chuo, 1-1-1, Higashi, Tsukuba, Ibaraki,        Japan)

The microorganisms may satisfactorily be microbial strains newlyseparated from the natural resources, such as in soil and from plants ormay satisfactorily be microbial strains artificially prepared bytreatment with mutagenic chemicals and recombinant DNA technology.

In one of preferable embodiments of the present invention, an intendedgene encoding an enzyme catalyzing the intended transamination from thesubstituted α-keto acid to glutamate derivatives may be integrated intomicrobial cells as well. Numerous examples for preparing useful proteinssuch as enzyme and physiologically active substances using recombinantDNA technology have been known. The use of recombinant DNA technologyenables mass preparation of useful proteins from natural origins at atrace amount. The gene to be integrated includes L-amino acidtransaminase genes and D-amino acid transaminase genes. One possibleexample is the introduction of D-amino acid transaminase genes fromBacillus sphaericus or Bacillus macerans into microorganisms.

European Patent Publication 0 736 604 and Taylor, et al., Journal ofBacteriol., 1998, Vol. 180, No. 16, p. 4319 report about the D-aminoacid transaminase gene derived from Bacillus sphaericus.

As the D-amino acid transaminase gene derived from Bacillus macerans,additionally, the DNA of the D-amino acid transaminase gene derived fromBacillus macerans may be used, which is described as SEQ ID No. 17 inthe sequence listing. When the DNA of the D-amino acid transaminase genederived from Bacillus macerans as described as SEQ ID No. 17 in thesequence listing is used, the D-amino acid transaminase described as SEQID No. 18 in the sequence listing may be obtained. Herein, the geneencoding the D-amino acid transaminase from Bacillus macerans and theamino acid sequence thereof are first elucidated by the inventors.

The origin of the D-amino acid transaminase gene is not limited to it.Any gene encoding D-amino acid transaminase generating an intendedD-glutamate derivative may be satisfactory.

In case of mass production of a protein using recombinant DNAtechnology, microbial cells, actinomycetes cells, yeast cells, fungalcells, plant cells, animal cells or the like may be used as the hostcells to be transformed. Among them, microorganisms, for which theknowledge with recombinant DNA technology exists, include for exampleBacillus, Pseudomonas, Brevibacterium, Corynebacterium, Streptomyces andEscherichia coli. Owing to numerous findings about the technique formass production of protein using bacteria of Escherichia, bacteria ofEscherichia are generally used. Preferably, Escherichia coli is used.

Using vectors such as plasmid or phage carrying the intended gene of atransaminase, the gene may satisfactorily be introduced into thesemicroorganisms. Otherwise, the intended gene may be integrated into thechromosome of the cell by homologous recombination. So-called multi-copyplasmid vectors are preferable and include for example plasmids with anorigin of replication as derived from Col E1 as the vector forEscherichia coli, which are for example pUC-base plasmids, pBR322-baseplasmids or derivatives thereof. For these vectors, promoters forgeneral use in protein production in Escherichia coli may be used as thepromoter for expressing the intended transaminase gene and includestrong promoters for example T7 promoter, trp promoter, lac promoter,tac promoter and PL promoter. To increase the productivity, a terminatoras a transcription termination sequence is preferably conjugated to thedownstream of the protein gene. Such terminator includes for example T7terminator, fd phage terminator, T4 terminator, terminators fortetracycline resistant gene, and terminators for Escherichia coli trp Agene. Additionally for screening transformants, preferably, the vectorhas a marker such as ampicillin resistant gene. As such plasmids,expression vectors with strong promoters are commercially available,such as pUC series (manufactured by TAKARA BIO INC.), pPROK series(manufactured by Clontech Laboratories, Inc.), and pKK233-2(manufactured by Clontech Laboratories, Inc.).

For a method for culturing a microorganism generating an enzyme for usein reaction 3, culture media for general use in the field, namelyculture media containing carbon sources, nitrogen sources, inorganicsalts, trace metal salts, and vitamins may be used. Depending on thetype of a microorganism or the culture conditions, about 0.1 to 1.0 g/dlof amino compounds such as amino acid is added to such culture medium,to promote the transamination activity.

When culturing genetic recombinant cells, chemicals such as ampicillin,kanamycin, neomycin and chloramphenicol may be appropriately added in amanner depending on the selected marker for the vector. Depending on thepromoter carried on the vector, the expression level of the recombinantgene may be raised by adding an appropriate amount of an inductionagent. In case of conjugating an intended gene to the downstream of thelac promoter to construct a vector, in one example, isopropyl1-thio-β-D-galactopyranoside (IPTG) is possibly added at an amount to afinal concentration range of 0.1 mM to 5 mM. Instead, galactose may beadded appropriately to a final concentration of 0.1 to 5 g/dl,preferably 0.5 g/dl to 2 g/dl.

As substances for use as the culture medium components, for example, anycarbon source that is compatible with the microorganism to be used maybe satisfactory with no specific limitation. For example, glucose,sucrose, fructose, glycerol and acetic acid or mixtures of them may beused. As the nitrogen source, ammonium sulfate, ammonium chloride, urea,yeast extract, meat extract, corn steep liquor, and casein hydrolyzedproducts or mixtures of them may be used. As a culture mediumcomposition, for example, a culture medium containing 0.5 g/dl fumaricacid, 1 g/dl yeast extract, 1 g/dl peptone, 0.3 g/dl ammonium sulfate,0.3 g/dl K₂HPO₄, 0.1 g/dl KH₂PO₄, 1 mg/dl FeSO₄.7H₂O, and 1 mg/dlMnSO₄.4H₂O, pH 7.0 is listed.

The culture temperature is generally within a range where microorganismsused may grow, namely a range of 10 to 45° C. The temperature ispreferably within a range of 20 to 40° C., more preferably within arange of 25 to 37° C. The pH of the culture medium is adjusted to arange of preferably 2 to 12, more preferably 3 to 10, still morepreferably 4 to 8. The aeration conditions are set to conditionssuitable for the growth of microorganisms used. Aerobic conditions arepreferable. The culture period is generally about 12 to 120 hours,preferably about 24 to 96 hours.

(C-2) Reaction Conditions for Reaction 3

The reaction 3 characteristically produces glutamate derivatives of thegeneral formula (2) from the substituted α-keto acid represented byformula (1).

The term “in the presence of an enzyme” for reaction 3 means that theenzyme should exist at its state to enable the generation of glutamatederivatives of formula (2) from the substituted α-keto acid representedby formula (1) in the reaction system. In other words, the enzyme mayexist at any state in the reaction system as long as the enzyme mayconvert the substituted α-keto acid represented by formula (1) to theglutamate derivative general formula (2). For example, the enzyme maysingly be added to the reaction system.

Otherwise, a microorganism with the enzyme activity (e.g. microorganismgenerating the enzyme, cells transformed with recombinant DNA, etc.), aculture of the microorganism (e.g., liquid culture, solid culture,etc.), a culture medium (e.g., cultures from which microbial cells arepreliminarily eliminated), and treated products of the culture may alsobe added to the reaction system. When using cultures of microorganisms,reaction 3 may be progressed concurrently with culturing themicroorganisms, or reaction 3 may be performed using culturespreliminarily prepared for obtaining the enzyme.

Herein, the term “treatment” means treatment for the purpose ofrecovering the enzyme in microbial cells and includes for exampletreatments with ultrasonication, glass bead, French press, andfreeze-drying and treatments with lysed enzyme, organic solvents,detergents and the like. The treated products after these treatments maybe further treated by routine processes (liquid chromatography, ammoniumsulfate and the like), to recover crude fractionated enzymes or purifiedenzymes. When these enzymes have the required ability, they also may beused.

Furthermore, the culture or treated product when used may be included incarrageenan gel or polyacrylamide or may be immobilized on films ofpolyether sulfone, regenerated cellulose and the like.

In reaction 3, the substituted α-keto acid as the substrate includes forexample the substituted α-keto acid represented by formula (1).

The reaction system may satisfactorily contain coenzymes, detergents,organic solvents and the like to accelerate the reaction. So as toincrease the permeability of the substituted α-keto acid as thesubstrate into microbial cells, for example, detergents such as Triton Xand Tween and organic solvents such as toluene and xylene may also beused. Further, coenzymes such as pyridoxal-5-phosphate may also be addedto the culture medium.

When dividing the culture for the generation of the enzyme and thereaction 3 and then sequentially carrying out these steps, the latterreaction 3 step is not necessarily done in an aerobic atmosphere. In ananaerobic atmosphere, rather, reaction 3 may be performed in a reactionsystem from which dissolved oxygen in the reaction solution is removedwith nitrogen gas substitution, argon gas substitution and sodiumsulfite addition.

The reaction temperature is generally within a temperature range wherethe enzyme used may be active, preferably within a range of 10 to 50°C., more preferably within a range of 20 to 40° C., and still morepreferably within a range of 25 to 37° C. The pH of the reactionsolution is adjusted to a range of generally 2 to 12, preferably 6 to 11and more preferably 7 to 9. The reaction time is generally about 1 to120 hours, preferably about 1 to 72 hours and more preferably about 1 to24 hours.

When determining the glutamate derivative or the substituted α-keto acidquantities in the liquid culture or the reaction solution, further, theglutamate derivative or the substituted α-keto acid may be assayedimmediately using well-known methods. For simple procedure, thin layerchromatography using “Silica gel 60F254” manufactured by Merck Ltd. maybe used. For enhancing the analytical precision, high performance liquidchromatography (HPLC) utilizing optical resolution columns such as“Inertsil ODS-80A” manufactured by GL Sciences, Inc. and “CROWNPAK CR(+)” manufactured by Daicel Chemical Industries, Ltd. may be used. Insuch manner, the glutamate derivative accumulated in the liquid cultureor the reaction solution may be collected from the liquid culture or thereaction solution by routine methods, prior to use. For the collectionfrom the liquid culture or the reaction solution, an appropriatecombination of well-known measures for general use in this field, forexample procedures such as filtration, centrifugation, vacuumconcentration, ion exchange chromatography, adsorption chromatographyand crystallization may be used.

The intended glutamate derivative may be obtained in the free form. Ifnecessary, the glutamate derivative may also be recovered in a salt formthereof. The salt form includes salts thereof with bases. For example,inorganic bases such as sodium hydroxide, potassium hydroxide andcalcium hydroxide and organic bases such as ammonia and various aminesare listed.

The above written description of the invention provides a manner andprocess of making and using it such that any person skilled in this artis enabled to make and use the same, this enablement being provided inparticular for the subject matter of the appended claims, which make upa part of the original description.

As used herein, the phrases “selected from the group consisting of,”“chosen from,” and the like include mixtures of the specified materials.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and subranges within a numerical limit orrange are specifically included as if explicitly written out.

The above description is presented to enable a person skilled in the artto make and use the invention, and is provided in the context of aparticular application and its requirements. Various modifications tothe preferred embodiments will be readily apparent to those skilled inthe art, and the generic principles defined herein may be applied toother embodiments and applications without departing from the spirit andscope of the invention. Thus, this invention is not intended to belimited to the embodiments shown, but is to be accorded the widest scopeconsistent with the principles and features disclosed herein.

Having generally described this invention, a further understanding maybe obtained by reference to certain specific examples, which areprovided herein for purposes of illustration only, and are not intendedto be limiting unless otherwise specified.

EXAMPLES

The invention is now described in detail in the following examples, butthe invention is not limited to only these examples.

Example 1

Example 1 relates to reaction 1 of the invention. In Example 1,L-tryptophan, indole-3-pyruvic acid and indoleacetic acid are measuredby high performance liquid chromatography (column: Inertsil ODS-2(4.6×250 mm); column temperature: 40° C.; eluate: 0.1 M KH₂PO₄—H₃PO₄(pH=2.80)/CH₃CN=1/9 to 5/5; flow rate of 1.0 ml/min; detection: UV 210nm).

(1-1) Generation of indole-3-pyruvic acid from L-Trp Via the Reaction ofMicrobial Cells with Amino Acid Oxidase Activity

50 ml of a culture medium, pH 7.0 containing 1 g/dl yeast extract, 1g/dl polypeptone, 0.3 g/dl (NH₄)₂SO₄, 0.3 g/dl K₂HPO₄, 0.1 g/dl KH₂PO₄,0.05 g/dl MgSO₄.7H₂O, 1 mg/dl FeSO₄.7H₂O, and 1 mg/dl MnSO₄.4H₂O areplaced in a 500-ml Sakaguchi flask for sterilization at 110° C. for 10minutes.

Innocluate a culture medium with one loop of Achromobacter sp. AJ2425,Proteus rettgeri or Morganella morganii preliminarily cultured in abouillon agar culture medium at 30° C. for 24 hours. Culture withagitation at 30° C. for 24 hours. After culturing, harvest microbialcells from the culture by centrifugation, individually rinse in 50 ml of20 mM Tris-HCl buffer, pH 7.6, and prepare as rinsed microbial cells,again by centrifugation.

Add the wet microbial cells to a reaction solution of 1 g/dlL-tryptophan and 20 mM Tris-HCl buffer, pH 8.0 to a wet microbial cellweight of 1 w/v %. Transfer 1 ml of the reaction solution into a 5-mltest tube, shake at 30° C. for one hour to facilitate the productconversion. After the completion of the reaction, measure the amount ofgenerated indole-3-pyruvic acid, the amount of residual L-tryptophan(L-Trp) and the amount of the by-product indoleacetic acid (IAA) (seeTable 1).

TABLE 1 Amount of indole pyruvic acid generated from L-tryptophanStrains L-Trp (g/dl) IPA (g/dl) IAA (g/dl) Achromobacter sp. AJ2425 0.020.97 0.03 Proteus rettgeri IFO13501 0 0.98 0.03 Morganella morganiiIFO3168 0 0.99 0.02

Consequently, indole-3-pyruvic acid is accumulated in any of theexperimental lots where the reaction with the rinsed microbial cells isperformed. Thus, indole-3-pyruvic acid is almost quantitativelygenerated from 1 g/dl L-tryptophan.

(1-2) Recovery of indole-3-pyruvic acid by Nitrogen Gas SubstitutionTreatment of the Reaction Solution with the Rinsed Microbial Cells ofMorganella morganii IFO3168 and Crystallization in Hydrochloric Acid(a) Preparation of the Reaction Solution with the Rinsed Microbial Cellsof Morganella morganii

By the same method as in (1-1), the rinsed microbial cells of Morganellamorganii were prepared. Six Sakaguchi's flasks containing 50 ml of thereaction solution of 1 g/dl L-tryptophan and 20 mM Tris-HCl buffer, pH8.0 were prepared. The wet microbial cells prepared were added to theindividual flasks to a wet microbial cell weight of 1 w/v %, forreaction under shaking at 30° C. for one hour. After the completion ofthe reaction, the microbial cells were removed by centrifugation, toobtain the reaction solution of about 290 ml.

(b) Recovery of indole-3-pyruvic acid by Nitrogen Gas Substitution ofthe Reaction Solution and Acid Crystallization

74 ml of the reaction solution obtained in (a) were transferred into around-bottom flask for nitrogen gas substitution. Hydrochloric acid wasadded so as to adjust the reaction solution to pH 2 or less. 15 ml of 6Nhydrochloric acid were added to 74 ml of the reaction solution (to afinal hydrochloric acid concentration of about 1 N), and the mixture isstirred at 20° C. Through the procedure, the crystal was deposited. 24hours later, the mixture was filtered. The resulting crystal was rinsedin 15 ml of water. The wet crystal thus obtained was dried under reducedpressure at 40° C., and obtained indole-3-pyruvic acid was 684 mg (yieldof 79.5% from the starting tryptophan). The resulting indole-3-pyruvicacid was a yellowish white crystal, and the content was 97.2 wt % byhigh performance liquid chromatography (HPLC).

(c) Recovery of indole-3-pyruvic acid by Acid Crystallization ofReaction Solution

66 ml of the reaction solution obtained in (a) were transferred into around-bottom flask. 6N Hydrochloric acid of 13 ml was added so as toadjust the reaction solution to pH 2 or less and stirred at 20° C.Through the procedure, the crystal was deposited. 24 hours later, themixture was filtered. The resulting crystal was rinsed in 13 ml ofwater. The wet crystal thus obtained was dried under reduced pressure at40° C., to obtain indole-3-pyruvic acid at 538 mg (yield of 58.2% fromthe starting tryptophan). The resulting indole-3-pyruvic acid was a darkbrown crystal, and the content was 80.5 wt % measured by highperformance liquid chromatography (HPLC).

(d) Comparison of the Obtained indole-3-pyruvic acid

The indole-3-pyruvic acid (IPA) thus obtained in (b) was compared withthat obtained in (c) in terms of crystal quality (see Table 2).

As apparently shown from these results, the lot (b) having involvednitrogen gas substitution included a higher IPA content and a reducedcontent of the by-product indoleacetic acid (IAA) as an impurity in thecrystal. Further, the coloring of the resulting crystal was suppressedin the lot having involved nitrogen gas substitution. The crystals inthe individual lots was diluted to 10 mg/dl, of which the transmittanceat 450 nm and 400 nm was then measured. It was confirmed that thetransmittance in the nitrogen gas substitution lot was reduced, i.e. thecoloring via decomposition was suppressed.

TABLE 2 IPA crystal quality With nitrogen Without substitution (b)nitrogen substitution (c) IPA content in crystal 97.30% 80.50% IAAcontent in crystal  0.18%  1.54% Crystal color yellowish white darkbrown Transmittance (450 nm)  96.9% T  82.9% T Transmittance (400 nm) 94.1% T  75.9% T

As apparently shown in the results, indole-3-pyruvic acid mayefficiently be produced from tryptophan in a simple manner.

Example 2

In Example 2, the reaction 2 is performed using a chemical syntheticsystem.

(2-1) Synthesis of 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid(IHOG)

7.50 g of indole-3-pyruvic acid (35.8 mmol; content of 97.0% by weight)and 14.18 g of oxaloacetic acid (107.4 mmol) are added to and dissolvedin 64.45 ml of water preliminarily dissolving 18.91 g of potassiumhydroxide (286.5 mmol; content of 85% by weight) therein. The mixturesolution is agitated at 35° C. for 24 hours.

Further, 40.0 ml of 3N hydrochloric acid is added for neutralization (pH7.0), to obtain a neutralized reaction solution of 153.5 g. Theneutralized reaction solution contained 5.55 g of IHOG, so the yield is53.3% (vs. indole-3-pyruvic acid).

Water is added to the neutralized reaction solution to 168 ml and ispassed through a resin column (diameter of 4.8 cm) packed with 840 ml ofa synthetic adsorbent (DIAION-SP207 manufactured by Mitsubishi ChemicalCorporation). Further, pure water is passed through the column at a flowrate of 23.5 ml/minute, to collect 1.73 to 2.55 (L/L-R) to obtain anaqueous solution containing 3.04 g of IHOG at high purity at a yield of54.7% (vs. the charged amount to the resin).

(Nmr Measurement)

¹H-NMR (400 MHz, D₂0): 3.03 (d, 1H, J=14.6 Hz), 3.11 (d, 1H, J=14.6 Hz),3.21 (d, 1H, J=18.1 Hz), 3.40 (d, 1H, J=18.1 Hz), 7.06-7.15 (m, 3H),7.39 (d, 1H, J=7.8 Hz), 7.66 (d, 1H, 7.8 Hz).

¹³C-NMR (100 MHz, D₂O): 35.43, 47.91, 77.28, 109.49, 112.05, 119.44,119.67, 121.91, 125.42, 128.41, 136.21, 169.78, 181.43, 203.58

(2-2) Synthesis of 4-phenylmethyl-4-hydroxy-2-oxoglutaric acid (PHOG)

5.0 g (30.5 mmol) of phenylpyruvic acid and 12.1 g (91.4 mmol) ofoxaloacetic acid are added to 25 ml of water preliminarily dissolving13.8 g of potassium hydroxide (purity of 85%) therein, for reaction atambient temperature for 72 hours. Using the conc. hydrochloric acid, thereaction solution is adjusted to pH 2.2, for extraction into ethylacetate. The organic layer is rinsed with saturated aqueous sodiumchloride, dried over anhydrous magnesium sulfate and concentrated toobtain the residue. The residue is recrystallized in ethyl acetate andtoluene to obtain PHOG of 2.8 g (11.3 mmol.) in crystal.

(Nmr Measurement)

¹H NMR (D₂ 0) δ: 2.48 (d, J=14.4 Hz, 0.18H) 2.60 (d, J=14.4 Hz, 0.18H),2.85-3.30 (m, 3.64H), 7.17-7.36 (m, 5H)

(Measurement of Molecular Weight)

Theoretical value by ESI-MS: C₁₂H₁₂O₆=252.23Experimental value: 251.22 (MH⁻)

Example 3

In example 3, reaction 2 is performed using an enzymatic system. InExample 3, herein, IHOG and PHOG used as substrates are syntheticallyprepared by the method described in Example 2.

(3-1) Screening of Microorganism with the Activity of Aldolase for PHOG(Referred to as PHOG Activity Hereinafter)

Screening of microbial strains having the aldolase activity isperformed, wherein 4-phenylmethyl-4-hydroxy-2-oxoglutaric acid (PHOG) isused as a substrate.

Test microorganisms (bacteria and yeast) are inoculated on a bouillonplate culture medium (Eiken Chemical Co., Ltd.), for culturing at 30° C.for 24 hours. The resulting culture is inoculated on a plate containing0.5 g/dl glycerol, 0.5 g/dl fumaric acid, 0.3 g/dl yeast extract, 0.2g/dl peptone, 0.3 g/dl ammonium sulfate, 0.3 g/dl K₂HPO₄, 0.1 g/dlKH₂PO₄, 0.05 g/dl MgSO₄.7H₂O, 0.25 g/dl sodium phthalate, and 2 g/dlagar powder (pH 6.5), for culturing at 30° C. for 24 hours. Theresulting microbial cells are then inoculated in a reaction solution of100 mM Tris-HCl, pH 8.0, 50 mM PHOG, 1 mM MgCl₂, 5 mM potassiumphosphate solution (KPi) and 1 v/v % toluene to a wet microbial cellweight of about 1 w/v %, for reaction at 30° C. for 24 hours.

The concentration of the free pyruvic acid in the reaction solution ismeasured by an enzymatic method using lactate dehydrogenase (LDH). 10 μLof sample is added to 200 μL of a reaction solution of 100 mM Tris-HCl,pH 8.0, 1.5 mM NADH, 5 mM MgCl₂, and 25 U/ml LDH, for incubation at 30°C. for 10 minutes. The absorbance at 340 mM is measured after thereaction, to determine the amount of pyruvic acid in the sample, basedon a reduction in the amount of NADH.

Additionally, the amount of the phenylpyruvic acid generated is assayedby HPLC analysis using “Inertsil ODS-2” (5 μm, 4.6×250 mm) manufacturedby GL Sciences, Inc. The analytical conditions are as follows.

Mobile phase: 20 v/v % acetonitrile/aqueous 0.05 v/v % trifluoroaceticacid solution

Flow rate: 1 ml/minColumn temperature: 40° C.

Detection: UV 210 nm.

Under these conditions, PHOG is eluted at a retention time of about 9.8minutes, while phenylpyruvic acid is eluted at a retention time of about12 minutes. These are individually fractionated and assayed.

The value of the amount of pyruvic acid or phenylpyruvic acid generatedfrom PHOG in the test microbial cell-added lot minus the amount thereofin the control lot (no microbial cell-added lot) is defined as theamount thereof generated with aldolase. Consequently, the aldolaseactivity for the substrate PHOG in the microbial strains is shown inTable 3.

TABLE 3 Screening results of microbial strains with PHOG aldolaseactivity Pyruvic acid Phenylpyruvic Strains (mM) acid (mM) Pseudomonastaetrolens ATCC4683 34.9 35.0 Pseudomonas coronafaciens AJ2791 33.6 33.9Pseudomonas desmolytica AJ1582 1.1 2.9 Erwinia sp. AJ2917 0.8 3.0Flavobacterium rhenanum AJ2468 3.0 6.1 Xanthomonas citri AJ2797 1.0 3.2

Pseudomonas taetrolens ATCC4683 is selected, and the PHOG syntheticreaction from phenylpyruvic acid and oxaloacetic acid or pyruvic acid isexamined. The microbial cells of P. taetrolens ATCC4683 (AJ2212) areinoculated in a reaction solution of 100 mM Tris-HCl, pH 8.0, 50 mMphenylpyruvic acid, 1 mM MgCl₂, 5 mM KPi, 100 mM oxaloacetic acid orpyruvic acid and 1 w/w % toluene to a final concentration of about 1 w/v%, for reaction at 30° C. for 16 hours. After the completion of thereaction, the amount of PHOG generated is assayed by HPLC. The amount ofPHOG generated from phenylpyruvic acid and oxaloacetic acid or pyruvicacid is shown in Table 4.

TABLE 4 Amount of PHOG generated from phenylpyruvic acid and oxaloaceticacid or pyruvic acid Oxaloacetic acid lot Pyruvic acid lot Microbialcell-added lot 14.3 mM 9.3 mM Control lot (Mg added) 8.6 1.7 Control lot(no Mg added) Trace N.D.

Table 4 shows that the amount of PHOG generated in the microbialcell-added lot increased and that the activity of aldolase may generatePHOG from any combinations of phenylpyruvic acid+oxaloacetic acid and ofphenylpyruvic acid+pyruvic acid.

(3-2) Purification of Aldolase Derived from Pseudomonas taetrolensATCC4683 for IHOG

Aldolase for IHOG (sometimes referred to as IHOG aldolase hereinafter)is purified as follows from a soluble fraction of the P. taetrolensstrain ATCC4683. As to the assaying of the aldolase activity, the aldoldecomposition (retroaldol) activity using PHOG as the substrate ismeasured under the following conditions.

Reaction conditions: 50 mM Tris-HCl, pH 8.0, 2 mM PHOG, 0.2 mM NADH, 0.2mM KPi, 1 mM MgCl₂, 16 U/ml lactate dehydrogenase, and 3 μl enzyme/600μl reaction solution, for the measurement of absorbance at 30° C. and340 nm.

1. Preparation of Soluble Fraction:

One loop of the microbial cells of P. taetrolens ATCC4683 preliminarilycultured in a bouillon plate culture medium at 30° C. for 24 hours isinoculated in a 500-ml flask containing 50 ml of an enzyme-generatingculture medium (0.5 g/dl glycerol, 0.5 g/dl fumaric acid, 0.5 g/dlammonium sulfate, 0.3 g/dl K₂PO₄, 0.1 g/dl KH₂PO₄, 0.05 g/dl MgSO₄.7H₂O,0.3 g/dl yeast extract, 0.2 g/dl peptone, 0.25 g/dl sodium phthalate,and 0.005% Antifoam A (manufactured by Sigma). After adjustment to pH6.5 with KOH), the culture is incubated with agitation at 30° C. for 24hours.

0.5 ml of the liquid culture is inoculated in 40 flasks of a 500-mlvolume, each of the flasks containing 50 ml of the enzyme-generatingculture medium, for culturing under shaking at 30° C. for 24 hours. Themicrobial cells are harvested from the resulting liquid culture bycentrifugation, and suspended and washed in buffer A (20 mM Tris-HCl, pH7.6), followed by centrifugation again for harvesting the microbialcells. The resulting washed microbial cells are suspended in 200 ml ofbuffer A, for ultrasonic disruption at 4° C. for 30 minutes. Thesolution after the disruption is centrifuged (×8,000 rpm, 10 minutes×twotimes) to remove the residual microbial cells, followed by additionalultra-centrifugation (×50,000 rpm, 30 minutes) to recover the resultingsupernatant, which is defined as soluble fraction.

2. Anion Exchange Chromatography: Q-Sepharose FF

80 ml of the soluble fraction is treated in an anion exchangechromatography column Q-Sepharose FF 26/10 (manufactured by Pharmacia,CV=20 ml), for adsorption onto the carrier. Proteins that did not adsorbonto the carrier (non-adsorbed proteins) are washed out, using Buffer A.Subsequently, the adsorbed protein is eluted while the KCl concentrationis changed linearly from 0 M to 0.7 M (in total of 140 ml). By detectingthe activity of aldolase for PHOG (sometimes referred to as PHOGaldolase hereinafter) in each of the eluted fractions, the PHOG aldolaseactivity peak is detected in the fraction corresponding to about 0.5 M.The same chromatographic procedures are repeatedly carried out for twotimes.

3. Hydrophobic Chromatography: Phenyl Sepharose HP HR 16/10

The solution with the detected aldolase activity is collected anddialyzed with Buffer B (50 mM Tris-HCl, pH 7.6, 1M ammonium sulfate, pH7.6) at 4° C. overnight and then filtered through a 0.45-μm filter. Theresulting filtrate is treated with a hydrophobic chromatography columnPhenyl Sepharose HP HR 16/10 (manufactured by Pharmacia) equilibratedwith Buffer B. Through the procedures, the aldolase is adsorbed on thecarrier.

The non-adsorbed proteins that had not been adsorbed on the carrier arewashed out, using Buffer B. Subsequently, the aldolase is eluted whilelinearly changing the ammonium sulfate concentration from 1M to 0 M. Bymeasuring the aldolase activity in each of the eluted fractions, thealdolase activity peak is detected in the fraction corresponding toabout 0.2 M of the ammonium sulfate concentration.

4. Gel Filtration Chromatography: Sephadex 200 HP 16/60

Individual fractions containing aldolase are combined and dialyzed withBuffer A, and filtered through a 0.45-μm filter. The resulting filtrateis concentrated with an ultrafiltration membrane centriprep 10 and,subsequently is treated with a gel filtration Sephadex 200 HP 16/60(manufactured by Pharmacia) equilibrated with Buffer C (20 mM Tris-HCl,pH 7.6, 0.1 M KCl) for elution at a flow rate of 1 ml/min. Through theprocedures, the aldolase is eluted in fractions from 66 to 71 ml. Basedon the position of the activity peak eluted, it is estimated that themolecular weight of the aldolase would be about 146 kDa.

5. Anion Exchange Chromatography: Mono Q HR5/5

The resulting fractions are filtered through a 0.45-μm filter. Theresulting filtrate is treated with an anion chromatography column Mono-QHR 5/5 (manufactured by Pharmacia) equilibrated with Buffer A. Throughthe procedures, the aldolase is adsorbed on the carrier. Thenon-adsorbed proteins are washed out, using Buffer A. Subsequently, theprotein is eluted while the KCl concentration is linearly changed from 0mM to 700 mM (in total of 24 ml). By measuring the aldolase activity ineach eluted fractions, the aldolase activity peak is detected in thefraction corresponding to about 0.4 M of the KCl concentration.

6. Hydroxyapatite Chromatography: CHT-II

The resulting fraction is dialyzed with Buffer D (10 mM potassiumphosphate buffer, pH 7.0) at 4° C. overnight, and filtered through a0.45-μm filter. The resulting filtrate is treated with a hydroxyapatitechromatography column CHT-II 5 ml (manufactured by Bio-Rad LaboratoriesInc.) equilibrated with Buffer D. Through the procedures, the aldolasecan be separated from the adsorbed proteins because the aldolase is notadsorbed on the carrier.

The fraction purified by the aforementioned column chromatographicprocedures is treated with SDS-PAGE. An almost single band at a positioncorresponding to about 25 kDa is detected. Because it is estimated bygel filtration chromatography that the molecular weight is about 146kDa, it is speculated that the aldolase would be forming a hexamer.Table 5 shows purification tables.

TABLE 5 Purification table of Pseudomonas taetrolens strainATCC4683-derived IHOG aldolase Protein specific activity purificationtotal yield (mg) (U/mg) fold activity (U) (%) soluble fraction 37500.014 1 51 100 Q-sepharose HP 26/10 510 0.060 4.4 30.5 59.8 Phenylsepharose HP 16/10 21.2 0.893 66 19.0 37.2 Sephadex200 HP 16/60 1.94.643 341 8.65 17.0 monoQ HR5/5 0.49 10.89 800 5.33 10.4 HydroxyapatiteCHT-II 0.025 28.70 2110 0.71 1.4

(3-3) Determination of Internal Amino Acid Sequence of IHOG Aldolase

About 2 μg portion of the purified aldolase is treated for SDS-PAGE andseparated thereby. Subsequently, the sample in the SDS-PAGE gel istreated with trypsin (pH 8.5, 35° C., 20 hours) and treated byreverse-phase HPLC to separate fragmental peptides. The amino acidsequences of two of the separated fractions are determined as follows,which are composed of 20 residues and 12 residues as SEQ ID Nos. 4 and5, respectively.

TABLE 6 Internal amino acid sequence determined SQ ID No. 4SLLDA FQNVV TPHIS DNLGR SQ ID No. 5 AEIAT GALDQ SW(3-4) Cloning of the Gene of P. taetrolens Strain ATCC4683-Derived IHOGAldolase

1. Preparation of Chromosomal DNA

The P. taetrolens strain ATCC4683 is cultured in 50 ml of a bouillonculture medium at 30° C. overnight (pre-culture). 5 ml of the liquidculture is used as a seed bacterium, for culturing in 50 ml of abouillon culture medium. After culturing up to the latter logarithmicgrowth stage, 50 ml of the liquid culture is treated by centrifugation(12,000×g, 4° C., 15 minutes) to harvest the microbial cells. Using themicrobial cells, the chromosomal DNA is prepared by the routine method.

2. Identification of Internal Sequence by PCR

Based on the internal amino acid sequence of the determined IHOGaldolase, the following mix primer (SEQ ID Nos. 6 and 7) is prepared.

TABLE 7 Mix primer designed and synthesized on the basisof the internal amino acid sequence SQ ID No. 6TTY CAR AAY GTS GTS ACS CCS C SQ ID No. 7TGR TCR ATN GCN CCS GTN GCR ATY TCN GC

Using the prepared primer mix, PCR amplification is performed using thechromosomal DNA of P. taetrolens strain ATCC4683 as a template. The PCRis performed using PCR Thermal PERSONEL (manufactured by TAKARA BIOINC.) for 30 cycles under the following conditions:

-   -   94° C. for 30 seconds    -   55° C. for 30 seconds    -   72° C. for 1 minute.

PCR products are treated by agarose gel electrophoresis, so that afragment of about 500 bp is amplified. The DNA fragment is cloned inpUC18, for the determination of the nucleotide sequence. The amino acidsequence speculated on the basis of the recovered DNA fragment isidentical to the internal amino acid sequence of the IHOG aldolase, sothat the recovery of the intended aldolase gene is confirmed.

3. Cloning of the Full-Length Gene by Colony Hybridization

It is attempted to recover the full-length gene using the PCR-amplifiedDNA fragment by Southern analysis and colony hybridization. The DNAprobe is prepared using DIG High Prime (manufactured by RocheDiagnostics) according to the instruction manual, and then, the probe islabeled by overnight (O/N) incubation at 37° C.

Southern analysis is done by completely digesting 1 μg of thechromosomal DNA with various restriction enzymes, electrophoresis on0.8% agarose gel, blotting on nylon membrane and other proceduresfollowing the manual. Hybridization is performed using DIG Easy Hyb(manufactured by Roche Diagnostics), for pre-hybridization at 50° C. forone hour. Then, the probe is added for overnight hybridization. Thebands are detected, using DIG Nucleotide Detection Kit. Consequently, aPstI fragment of about 4 kbp is detected that strongly hybridized withthe PCR fragment as a probe.

Then, the PstI fragment is recovered by colony hybridization. 20 μg ofthe chromosomal DNA is treated with PstI and treated with agarose gelelectrophoresis, to recover a fragment of about 4 kbp. The fragment isconjugated into pUC118, to prepare a library in E. coli JM109. Thecolony is transferred onto a nylon membrane filter (Hybond-N,manufactured by Amersham), followed by alkali denaturation,neutralization and immobilization. Hybridization is performed, using DIGEasy Hyb. The filter is immersed in a buffer, for one-hourprehybridization at 42° C. Then, the prepared labeled probe is added,for hybridization at 42° C. for 16 hours. After rinsing in SSC, a colonyhybridizing with the probe is detected, using DIG Nucleotide DetectionKit (manufactured by Roche Diagnostics). Consequently, a clone stronglyhybridizing with the probe is obtained.

The nucleotide sequence of the plasmid DNA recovered from the resultingclone is determined. It is shown that the DNA had the nucleotidesequence described as SEQ ID No. 1. The 678-bp orf containing thenucleotide sequence (the 507-th to 566-th positions and the 1046-th to1082-th positions in SEQ ID No. 1) is determined, which corresponds tothe internal, determined amino acid sequence, and the intendedfull-length aldolase is obtained.

4. Expression of IHOG Aldolase in E. coli (NO. 1)

Using the primers (SEQ ID Nos. 8 and 9) shown in Table 8, the fragmentamplified from the chromosomal DNA of the P. taetrolens strain ATCC4683is treated with BamHI/HindIII and then inserted in the BamHI/HindIIIsite of pUC18, to construct a plasmid pUCALD. The constructed expressionplasmid is introduced in E. coli JM109. The resulting transformant isagitated in an LB culture medium containing 50 μg/ml ampicillin at 37°C. day and night (pre-culture). Then, the liquid pre-culture is seededat 1% in 50 ml of the LB culture medium, for culturing at 37° C. About 2hours after the start of culture, IPTG is added to a final concentrationof 1 mM, for additional 3-hour culture.

After the completion of culturing and expression of the gene undercontrol of an inducible promoter, the microbial cells are harvested andrinsed, and those are suspended in 1 ml of 20 mM Tris-HCl, pH 7.6. Themicrobial cells are then disrupted using Multi-Bead Shocker(manufactured by Yasui Kikai Corporation). The solution after thedisruption is centrifuged at 15,000 rpm for 10 minutes, so that theresulting supernatant is defined as crude enzyme solution.

TABLE 8 Primers SQ ID No. 8 ALD-5′ Bam(5′-GCC GGA TCC ACA AGG GTT CAG TCA TTC ATG G-3′) SQ ID No. 9ALD-3′ Hind(5′-CCG AAG CTT TCA GTT  CGC CAG GCC AGC C-3′)

Using the crude enzyme solution, the aldolase activity is measured usingthe substrate PHOG. While no PHOG aldolase activity is detected in E.coli harboring pUC18 (control), the PHOG aldolase activity of 0.81U/mg·protein is observed in the strain harboring pUCADL. This indicatesthat the gene encodes the intended aldolase.

(3-5) Synthesis of 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid(IHOG) from indole-3-pyruvic acid and Pyruvic Acid, Using the StrainExpressing Aldolase

The rinsed microbial cells of E. coli expressing aldolase as prepared in(3-4) are used as the enzyme source, for carrying out the synthesis of4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid (IHOG) fromindole-3-pyruvic acid and pyruvic acid. IHOG is quantitatively measuredby HPLC using “Inertsil ODS-2” (5 μm, 4.6×250 mm) manufactured by GLSciences, Inc. The analytical conditions are as follows.

-   -   Mobile phase: 40 v/v % acetonitrile/5 mM phosphate dihydrogen        tetrabutylammonium solution    -   Flow rate: 1 ml/min    -   Column temperature: 40° C.    -   Detection: UV 210 nm

The rinsed microbial cells of the aldolase-expressing E. coli are addedto a reaction solution of 100 mM buffer (Tris-HCl, pH 8.0 or pH 9.0 andglycine-NaOH pH 10.0), 50 mM indole-3-pyruvic acid, 250 mM pyruvic acid,1 mM MgCl₂, and 1 v/v % toluene to 10 w/v %, for reaction under shakingat 33° C. for 4 hours. The enzyme reaction solution is appropriatelydiluted, for measuring the resulting IHOG.

TABLE 9 Amount of IHOG generated with aldolase pH aldolase IHOG (mM) 8 +9.2 8 − 0.42 9 + 12.1 9 − 1.6 10 + 10.7 10 − 5.4

Consequently, the amount of generated IHOG is increased in thealdolase-expressing E. coli addition lot. Generation of IHOG by aldolaseis achieved.

(3-6) Mass Expression of IHOG Aldolase in E. coli (NO. 2)1. Construction of pTrp4 Plasmid Harboring trp Promoter and rrnBTerminator

Using the oligonucleotides in Table 10 as primers (a combination of SEQID Nos. 10 and 11), the promoter region in the trp operon on thechromosomal DNA of E. coli W3110 as the intended gene region isamplified by PCR. The resulting DNA fragment is ligated to thepGEM-Teasy vector (manufactured by Promega Ltd.). In the ligationsolution, E. coli JM109 is transformed, to facilitate selection of astrain having the intended plasmid where the trp promoter is insertedalong a direction inverse to the direction of the lac promoter among theresulting ampicillin resistant strains. Then, the plasmid is treatedwith Eco0109I/EcoRI, to obtain a DNA fragment containing the trppromoter, which is then ligated into the Eco0109I/EcoRI digestionproduct of pUC19 (manufactured by TAKARA BIO INC.). In the ligationsolution, E. coli JM109 is transformed, to facilitate selection of astrain having the intended plasmid among the resulting ampicillinresistant strains. The plasmid is designated as pTrp1.

Then, pKK223-3 (manufactured by Amersham Pharmacia) is treated withHindIII/HincII, to obtain a DNA fragment containing the rrnB terminator,which is then ligated to a HindIII/PvuII digestion product of pTrp1. Inthis ligation solution, E. coli JM109 is transformed to obtain a strainwith the intended plasmid among the resulting ampicillin resistantstrains. The plasmid is designated as pTrp2.

Subsequently, the oligonucleotides in Table 10 as primers (a combinationof SEQ ID Nos. 10 and 12) and pTrp2 as template are used for PCR toamplify the trp promoter region. The resulting DNA fragment is treatedwith Eco0109I/NdeI and ligated to the Eco0109I/NdeI digestion product ofpTrp2. In the ligation solution, E. coli JM109 is transformed, to selecta strain with the intended plasmid among the resulting ampicillinresistant strains. Then, the plasmid is designated as pTrp4.

TABLE 10 SQ ID  5′- GTATCACGAGGCCCTAGCTGTGGTGTCATGGTCGGTGATC No. 10 side       Eco0109I SQ ID  3′- TTCGGGGATTCCATATGATACCCTTTTTACGTGAACTTGCNo. 11 side             Nde I SQ ID  3′-GGGGGGGGCATATGCGACCTCCTTATTACGTGAACTTG No. 12 side         Nde I2. Construction of Aldolase Gene-Expressing Plasmids ptrpALD1 andptrpALD2 and Expression in E. coli

Using the primers shown in Table 11 (SEQ ID Nos. 9 and 13), a fragmentamplified from the chromosomal DNA of P. taetrolens strain ATCC4683 istreated with NdeI/HindIII and inserted into the NdeI/HindIII site ofpTrp4 to construct a plasmid ptrpALD1. The plasmid expresses thealdolase gene of the amino acid sequence SEQ ID No. 3 from the 444-thATG as the translation initiation codon in the nucleotide sequence SEQID No. 1. Additionally using the primers (SEQ ID Nos. 9 and 14), afragment amplified from the chromosomal DNA of P. taetrolens strainATCC4683 is treated with NdeI/HindIII and inserted into the NdeI/HindIIIsite of pTrp4, to construct a plasmid ptrpALD2. The resulting plasmidexpresses the aldolase gene of the amino acid sequence SEQ ID No. 2 fromthe 456-th ATG as the translation initiation codon in the nucleotidesequence SEQ ID No. 1.

The individually constructed expression plasmids are introduced in E.coli JM109. The resulting transformant is shaken in an LB culture mediumcontaining 50 μg/ml ampicillin at 37° C. day and night (pre-culture). 50ml of the liquid pre-culture is seeded at 1% in 50 ml of the LB culturemedium, for culturing at 37° C. About 2 hours after the start of theculture, IPTG is added to a final concentration of 1 mM, for additional3-hour culture. After the completion of the culture, the microbial cellsare harvested and rinsed, suspended in 1 ml of 20 mM Tris-HCl, pH 7.6,and disrupted with Multi-bead Shocker (manufactured by Yasui KikaiCorporation). The solution, after disruption, is centrifuged at 15,000rpm for 10 minutes, and the resulting supernatant is defined as crudeenzyme solution.

TABLE 11 Primers SQ ID No. 9 ALD-3′ Hind(5′-CCG AAG CTT TCA GTT  GGC CAG GCC AGC C-3′) SQ ID No. 13 ALD-5′ Nde-1(5′-GGT TCA GTC ACA TAT  GGA GGT CGC TAT GTC-3′) SQ ID No. 14ALD-5′ Nde-2(5′-ATG GAG GTC CAT TAG   TCA TTG CCC GGT TCA CGC-3′)

Using the crude enzyme solution, the aldolase activity is measured usingPHOG as the substrate. While no PHOG aldolase activity is detected in E.coli harboring pTrp4 (control), the PHOG aldolase activity of 16.1U/mg·protein is observed in the strain harboring ptrpADL1, while thePHOG aldolase activity of 36.0 U/mg·protein is observed in the strainharboring ptrpADL2. This indicates that the aldolase of SEQ ID No. 2 and3 have aldolase activity.

Example 4

Example 4 relates to reaction 3 of the invention. In Example 4, herein,monatin and 4-phenylmethyl-4-hydroxy-glutamic acid (PHG) are assayed byhigh performance liquid chromatography using “Inertsil ODS-80A” (5 μm,6×150 mm) manufactured by GL Sciences, Inc. The analytical conditionsare as follows.

Mobile phase: 12 v/v % acetonitrile/aqueous 0.05 v/v % trifluoroaceticacid solution

Flow rate: 1.5 ml/minColumn temperature: 30° C. and

Detection: UV 210 nm

Under the analytical conditions, (2S,4S)-monatin and (2R,4R)-monatinwith a retention time of 12.1 minutes, (2S,4R)-monatin and(2R,4S)-monatin with a retention time of 9.7 minutes, (2S,4S)—PHG and(2R,4R)—PHG with a retention time of 7.2 minutes, and (2S,4R)—PHG and(2R,4S)—PHG with a retention time of 6.0 minutes are fractionated andassayed.

If necessary, additionally, analysis by HPLC using an optical resolutioncolumn “CROWNPAK CR (+)” (4.6×150 mm) manufactured by Daicel ChemicalIndustries, Ltd. is done. The analytical conditions are as follows.

-   -   (In case of monatin)    -   Mobile phase: aqueous perchloric acid solution, pH 1.5/10 v/v %        methanol    -   Flow rate: 0.5 ml/min    -   Column temperature: 30° C. and    -   Detection: UV 210 nm

Under the analytical conditions, optical monatin isomers (2R,4S),(2R,4R), (2S,4R) and (2S,4S) are fractionated and assayed at retentiontimes of 42, 57, 64, and 125 minutes in this order.

-   -   (In case of PHG)    -   Mobile phase: aqueous perchloric acid solution, pH 1.5    -   Flow rate: 1 ml/min    -   Column temperature: 30° C. and    -   Detection: UV 210 nm

Under the analytical conditions, optical PHG isomers (2R,4S), (2R,4R),(2S,4R) and (2S,4S) at retention times of 20 minutes, 28 minutes, 31minutes and 46 minutes in this order may be fractionated and assayed.

(4-1) (2S,4S)-Monatin Production with L-Amino Acid Transaminase

Microorganisms shown below in Table 12 are inoculated on a bouillonplate culture medium (Eiken Chemical Co., Ltd.), and cultured at 30° C.for 24 hours. The microbial cells are inoculated in 1 ml of a reactionsolution of 100 mM Tris-HCl, pH 7.6, 30 mM IHOG, 100 mM L-glutamatemonosodium, 1 mM pyridoxal-5′-phosphate, and 0.5 (v/v) toluene to a wetmicrobial cell weight of 5% by weight, for incubation at 30° C. for 16hours. After the completion of the reaction, the monatin generatedthereby is assayed. This is shown in Table 12. (2S,4S)-Monatin could begenerated from IHOG.

TABLE 12 Strains Monatin generated (mM) Aeromonas hydrophila IFO3820 1.2Agrobacterium tumefaciens IFO3058 1.9 Alcaligenes faecalis ATCC8750 1.6Beijerinckia indica ATCC9037 0.2 Escherichia coli ATCC12814 0.6 Proteusrettgeri IFO13501 0.7 Morganella morganii IFO3848 1.2(4-2) (2S,4S)-PHG Production with L-Amino Acid Transaminase

Microorganisms shown below in Table 13 are inoculated on a bouillonplate culture medium (Eiken Chemical Co., Ltd.), for culturing at 30° C.for 24 hours. The microbial cells are inoculated in 1 ml of a reactionsolution of 100 mM Tris-HCl, pH 7.6, 30 mM PHOG, 100 mM L-glutamatemonosodium or L-aspartate monosodium, 1 mM pyridoxal-5′-phosphate, and0.5 (v/v) toluene to wet microbial cells weight of 5% by weight, forincubation at 30° C. for 16 hours. After the completion of the reaction,the generated PHG is assayed. This is shown in Table 13. (2S,4S)-PHGcould be generated from PHOG.

TABLE 13 PHG generated (mM) Strains L-Glu L-Asp Aeromonas hydrophilaIFO3820 8.9 8.9 Agrobacterium tumefaciens IFO3058 8.2 8.0 Alcaligenesfaecalis ATCC8750 4.9 10.7 Beijerinckia indica ATCC9037 7.4 2.7Escherichia coli ATCC12814 9.0 3.3 Proteus rettgeri IFO13501 10.2 9.0Morganella morganii IFO3848 10.4 5.2(4-3) (2S,4S)-PHG Production with L-Amino Acid Transaminase

Microorganisms shown below in Table 14 are inoculated on a bouillonplate culture medium (Eiken Chemical Co., Ltd.), and cultured at 30° C.for 24 hours. The culture medium containing 0.5 g/dl fumaric acid, 1g/dl yeast extract, 1 g/dl peptone, 0.3 g/dl ammonium sulfate, 0.3 g/dlK₂HPO₄, 0.1 g/dl KH₂PO₄, 1 mg/dl FeSO₄.7H₂O, and 0.1 g/dl MnSO₄.4H₂O, pH7.0 is divided in 50-ml portions in 500-ml Sakaguchi's flasks andsterilized at 110° C. for 10 minutes. Onto the resulting liquid culturemedium is inoculated one loop and is cultured with agitation at 30° C.for 16 hours.

1 ml of the liquid culture was centrifuged, to obtain microbial cells,which are then rinsed and harvested with 20 mM Tris-HCl, pH 7.6 and aresubsequently suspended in 1 ml of a reaction solution of 100 mMTris-HCl, pH 7.6, 50 mM PHOG, 100 mM monosodium L-glutamate, 1 mMpyridoxal-5′-phosphate and 0.5 v/v toluene. The resulting suspension istransferred in a 10-ml test tube, for reaction under shaking at 30° C.for 18 hours. The generated PHG is assayed after the completion of thereaction. This isshown in Table 14. (2S,4S)-PHG could be generated fromPHOG.

TABLE 14 Strains PHG generated (mM) Aeromonas hydrophila IFO3820 16.4Alcaligenes faecalis ATCC8750 12.3 Proteus rettgeri IFO13501 17.5Morganella morganii IFO3848 17.2(4-4) 2R-PHG Production with D-Amino Acid Transaminase

Microorganisms shown below in Table 15 are inoculated on a bouillonplate culture medium (Eiken Chemical Co., Ltd.), and cultured at 30° C.for 24 hours. This is inoculated in 1 ml of a reaction solutioncontaining 100 mM Tris-HCl, pH 7.6, 50 mM PHOG, 100 mM D-glutamic acid,100 mM D-alanine, 1 mM pyridoxal-5′-phosphate and 0.5 v/v toluene tofinal wet microbial cells weight of 5% by weight, for incubation at 30°C. for 16 hours. After the completion of the reaction, the generated PHGis assayed. This is shown in Table 15. (2R,4S)-PHG and (2R,4R)-PHG couldbe generated from PHOG.

TABLE 15 PHG generated (mM) Strains (2R,4R) (2R,4S) Bacillus sphaericusATCC10208 16.6 16.5 Bacillus pulvifaciens AJ1327 2.8 2.6 Paenibacilluslarvae subsp. pulvifaciens ATCC13537 3.0 2.8 Bacillus macerans AJ1617*7.1 7.0 Paenibacillus macerans ATCC8244 6.5 6.5 Bacillus lentus AJ126994.6 4.6 Bacillus lentus ATCC10840 4.2 4.3 *FERM P-18653(4-5) Preparation of E. coli Expressing DAT Derived from Bacillussphaericus (Referred to as BSDAT Hereinafter) and 2R-PHG Production byReaction with Rinsed Microbial Cells

1. Construction of Expression Plasmid

So as to express the D-amino acid transaminase gene (abbreviated as“bsdat” hereinafter) derived from Bacillus sphaericus in E. coli, aplasmid pUCBSDAT is constructed as follows, where the bsdat gene isconjugated to the downstream of the lac promoter of pUC18. Using thechromosomal DNA of the Bacillus sphaericus strain ATCC10208 as templateand oligonucleotides shown below in Table 16 as primers, first, the geneis amplified by PCR. In such manner, a DNA fragment corresponding to the8-th to 1278-th positions in the bsdat nucleotide sequence described asSEQ ID No. 2 in the text of European Patent publication 0 736 604 may beamplified. The fragment is treated with BamHI and PstI, conjugated tothe BamHI/PstI digestion product of pUC18, and introduced in E. coliJM109. From the resulting ampicillin resistant strains, a strain withthe intended plasmid is selected, to construct an expression plasmidpUCBSDAT.

TABLE 16 SQ ID No. 15 5′-CCG GGA TTC GTT AAT CCA AAC GTT  AGC TGSQ ID No. 16 5′-GGC CTC CAG TTA GGC ATT AAT TGA  AAT TGG2. Preparation of E. coli Expressing BSDAT

An E. coli transformant with pUCBSDAT is seed-cultured in an LB culturemedium (1 g/dl bacto-tryptone, 0.5 g/dl yeast extract, and 1 g/dl NaCl)containing 0.1 mg/ml ampicillin at 37° C. for 16 hours. 1 ml of the seedliquid culture is added to a 500-ml Sakaguchi's flask charged with 50 mlof the LB culture medium, and cultured at 37° C. 2.5 hours after thestart of the culture, isopropyl 1-thio-β-D-galactopyranoside (IPTG) isadded to a final 1 mM concentration, for additional 4-hours ofculturing. From the resulting liquid culture, the microbial cells areharvested and rinsed, to prepare E. coli expressing BSDAT.

3. Reaction with Rinsed Microbial Cells, Using E. coli Expressing BSDAT

The microbial cells prepared in above 2 are suspended in 1 ml of areaction solution containing 100 mM Tris-HCl, pH 7.6, 50 mM PHOG, 100 mMamino acid donors (D-Glu, D-Ala, L-Glu, L-Ala), 1 mMpyridoxal-5′-phosphate and 0.5 v/v % toluene to a final wet microbialcell weight of 5%, and the resulting suspension is transferred in a10-ml test tube, for reaction under shaking at 30° C. for 18 hours. ThePHG generated is assayed after the completion of the reaction. This isshown in Table 17. (2R,4R), (2R,4S) and (2S, 4S)-PHG could be generatedfrom PHOG.

TABLE 17 PHG (mM) generated via reaction with rinsed microbial cells,using E. coli expressing BSDAT Added amino acid donors Generated PHGD-Glu D-Ala L-Glu L-Ala (2R,4R) 20.7 25.1 N.D. 15.4 (2R,4S) 17.5 17.022.7  7.0 (2S,4S) trace trace 22.7 trace(4-6) Preparation of E. coli Expressing DAT Derived from Bacillusmacerans AJ1617 (Referred to as BMDAT Hereinafter) and 2R-MonatinProduction by Reaction with Rinsed Microbial Cells

1. Preparation of Chromosomal DNA

The Bacillus macerans strain AJ1617 is cultured overnight in a 50-mlbouillon culture medium at 30° C. (pre-culture). 5 ml of the liquidculture as a seed bacterium is cultured in a 50-ml bouillon culturemedium. After the microbial strain was cultured up to the latterlogarithmic growth stage, 50 ml of the liquid culture is treated by acentrifugation procedure (12,000×, 4° C., 15 minutes) for harvesting themicrobial cells. Using the microbial cells, the chromosomal DNA isprepared by the routine method.

2. Isolation of the Bacillus macerans-Derived D-Amino Acid TransaminaseGene (Referred to as bmdat Hereinafter) from Gene Libraries

First, one unit of a restriction enzyme EcoRI is added to 30 μg of thechromosomal DNA of the Bacillus macerans strain AJ1617, for 3-hourreaction at 37° C. for partial digestion. Then, fragments of 3- to 6 kbpare recovered from the DNA by agarose gel electrophoresis. Thesefragments are ligated to 1 μg of the EcoRI cleavage product of theplasmid pUC118 (after BAP treatment; manufactured by TaKaRa Brewery,Co., Ltd.), to transform E. coli JM109 to prepare gene libraries, whichare then plated on an LB culture medium (1% tryptone, 0.5% yeastextract, 1% sodium chloride, 2% agar, pH 7.0) containing ampicillin toform colonies. The developed colonies are cultured overnight in an LBliquid culture medium containing ampicillin andisobutyl-1-thio-β-D-galactopyranoside (IPTG) of 0.1 mM at 37° C., forcentrifugation to harvest the resulting microbial cells.

The resulting microbial cells are inoculated in a reaction solution of100 mM Tris-HCl, pH 8.0, 50 mM sodium pyruvate, 100 mM D-glutamic acid,1 mM pyridoxal-5′-phosphate and 1 v/v % toluene, for reaction at 30° C.for 30 minutes. After the completion of the reaction, the reactionsolution is centrifuged. 5 μl of the resulting separated supernatant isadded to a 96-well plate containing 200 μl of a reaction solution forpyruvic acid assay (100 mM Tris-HCl, pH 7.6, 1.5 mM NADH, 5 mM MgCl2, 16U/ml lactate dehydrogenase (manufactured by Oriental Yeast Co., Ltd.)),for reaction at 30° C. for 10 minutes.

Subsequently, the absorbance at 340 nm is read with a plate reader(SPECTRA MAX190, manufactured by Molecular Device). The same assay isconducted by adding sodium pyruvate to a final concentration of 0.2 mMto 1 mM. Using this as the standard, the amount of pyruvic acid reducedis assayed, to detect the D-amino acid transaminase activity.

Via the screening of clones with DAT activity, the clones with the DATactivity are collected. From these transformants, plasmids containingbmdat are prepared and defined as pUCBMDAT. The plasmid pUCBMDAT istreated with EcoRI and treated by agarose gel electrophoresis, so thatthe inserted fragment is estimated to be of a length of about 3.3 kbp.

3. Nucleotide Sequence of Inserted Fragment

The nucleotide sequence of the inserted fragment in the plasmid pUCBMDATis determined by dideoxy method. The ORF of about 850 bp correspondingto the 630-th to 1481-th positions in the sequence SEQ ID No. 17 in thesequence listing is found. The homology of the ORF to known sequences isexamined. The ORF is 91% homologus to the D-amino acid transaminase genederived from Bacillus sphaericus ATCC 10208 in terms of amino acidsequence and is 66% homologus to the D-amino acid transaminase genederived from Bacillus sp. YM-1 in terms of amino acid sequence and is42% homologus to the D-amino acid transaminase gene derived fromBacillus licheniformis ATCC 10716 in terms of amino acid sequence.

This clearly show that the ORF encoded the D-amino acid transaminasegenes. Herein, the homology is calculated using a gene analysis software“genetyx ver. 6” (GENETYX) while various parameters are used as theywere initially set.

4. Preparation BMDAT-Expressing E. coli

E. coli transformant with pUCBMDAT is seed-cultured in an LB culturemedium (1 g/dl bacto-tryptone, 0.5 g/dl yeast extract and 1 g/dl NaCl)containing 0.1 mg/ml ampicillin at 37° C. for 16 hours. 1 ml of the seedliquid culture is added to a 500-ml Sakaguchi's flask charged with 50 mlof the LB culture medium and cultured at 37° C. 2.5 hours after thestart of the culture, isopropyl 1-thio-β-D-galactopyranoside (IPTG) isadded to a final 1 mM concentration and cultured for additional 4-hour.From the resulting liquid culture, the microbial cells are harvested andrinsed, to prepare E. coli expressing BMDAT.

5. Reaction with Rinsed Microbial Cells, Using E. coli Expressing BMDAT

The microbial cells prepared above in 4 are suspended in 1 ml of areaction solution containing 100 mM Tris-HCl, pH 8.0, 50 mM IHOG, 200 mMD-alanine, 1 mM pyridoxal-5′-phosphate and 0.5 v/v % toluene to a finalwet microbial cell weight of 5%, and the resulting suspension istransferred in a 10-ml test tube, for reaction under shaking at 33° C.for 20 hours. The 2R-monatin generated is assayed after the completionof the reaction. Consequently, 22 mM 2R-monatin could be generated.

Numerous modifications and variations on the present invention arepossible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the accompanying claims, theinvention may be practiced otherwise than as specifically describedherein.

INDUSTRIAL APPLICABILITY

The process of producing monatin in accordance with the inventionutilizes a process of producing glutamate derivatives and may producemonatin very efficiently using tryptophan as a starting material in anenzymatic reaction. This process is very useful industrially,particularly in the field of food.

1. A method for producing 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaricacid comprising contacting indole-3-pyruvate with a polypeptide chosenfrom 4-hydroxy-4-methyl-2-oxoglutarate aldolase and4-hydroxy-2-oxoglutarate aldolase under conditions in which4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid is produced.
 2. Aprocess for producing monatin or a salt thereof comprising, underconditions in which monatin is produced: (a) providing tryptophan orsalt thereof, (b) contacting tryptophan or salt thereof with a firstpolypeptide that facilitates converting tryptophan or salt thereof toindole-3-pyruvic acid or a salt thereof, wherein said first polypeptideis selected from the group consisting of an L-tryptophan transaminase,an aspartate transaminase, an L-amino acid oxidase and a D-amino acidoxidase; (c) contacting said indole-3-pyruvic acid or a salt thereofwith a second polypeptide that facilitates converting indole-3-pyruvicacid or a salt thereof and oxaloacetic acid or a salt thereof or pyruvicacid or a salt thereof to 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaricacid or a salt thereof, wherein said second polypeptide is selected fromthe group consisting of 4-hydroxy-4-methyl-2-oxoglutarate aldolase and4-hydroxy-2-oxoglutarate aldolase; (d) contacting said4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof witha third polypeptide that facilitates converting4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof tomonatin or salt thereof, wherein said third polypeptide is selected fromthe group consisting of a tryptophan transaminase, a tyrosinetransaminase, a tryptophan dehydrogenase, an aspartate transaminase, aglutamate dehydrogenase, a phenylalanine dehydrogenase, a D-amino aciddehydrogenase, and a D-amino acid transaminase, thereby producingmonatin or a salt thereof.
 3. The method of claim 2, wherein at leastone of said first, second or third polypeptide is chosen from purifiedpolypeptides, polypeptides expressed from an exogenous microorganism,recombinant or isolated nucleic acid, chemically synthesizedpolypeptides, and polypeptides in recombinant cells or a culture ofrecombinant cells, and combinations thereof.
 4. The method of claim 2,wherein at least one of said polypeptides is provided in an unpurifiedculture of recombinant cells.
 5. The method of claim 4, wherein theunpurified culture of recombinant cells comprises an increased amount oftotal soluble protein content relative to the wild type recombinantcells.
 6. The method of claim 2, wherein the polypeptide is expressedfrom an exogenous, recombinant or isolated nucleic acid inside a cell,and wherein tryptophan or salt thereof is converted to monatin or saltthereof inside the cell.
 7. The method of claim 2, wherein the method isperformed in a single reaction system.
 8. The method of claim 7, furthercomprising providing an amino donor chosen from alanine, lysine, serine,glutamic acid, aspartic acid, cysteine, tryptophan, phenylalanine,methionine, tyrosine, isoleucine, leucine, valine, arginine, asparagine,glutamine, ornithine, histidine, and a combination thereof in step (d).9. The method of claim 7, wherein pyruvic acid or a salt thereof iscontacted with said second polypeptide and indole-3-pyruvic acid or asalt thereof in step (c).
 10. The method of claim 2, further comprisingpurifying said monatin or salt thereof.
 11. The method of claim 2, whereat least about 0.058 mg/mL of monatin or salt thereof is produced. 12.The method of claim 2, further comprising providing 2-ketoglutaric acidas an amino acceptor in step (b).
 13. The method of claim 2, whereinpyruvic acid or a salt thereof is contacted with said second polypeptideand indole-3-pyruvic acid or a salt thereof in step (c).
 14. A processfor producing monatin or a salt thereof comprising: (a) providingtryptophan or salt thereof, (b) contacting tryptophan or salt thereofwith a first polypeptide that facilitates converting tryptophan or saltthereof to indole-3-pyruvic acid or a salt thereof, wherein said firstpolypeptide is selected from the group consisting of an L-tryptophantransaminase, an aspartate transaminase, an L-amino acid oxidase and aD-amino acid oxidase; (c) contacting said indole-3-pyruvic acid or asalt thereof with a second polypeptide that facilitates convertingindole-3-pyruvic acid or a salt thereof and oxaloacetic acid or a saltthereof or pyruvic acid or a salt thereof to4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof,wherein said second polypeptide is selected from the group consisting of4-hydroxy-4-methyl-2-oxoglutarate aldolase and a4-hydroxy-2-oxoglutarate aldolase; (c) chemically reactingindole-3-pyruvic acid or a salt thereof with oxaloacetic acid or a saltthereof or pyruvic acid or a salt thereof under conditions in which analdol condensation occurs to produce4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof; (d)purifying 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a saltthereof; (e) contacting said4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof witha third polypeptide that facilitates converting4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof tomonatin or salt thereof, wherein said third polypeptide is selected fromthe group consisting of a tryptophan transaminase, a tyrosinetransaminase, a tryptophan dehydrogenase, an aspartate transaminase, aglutamate dehydrogenase, a phenylalanine dehydrogenase, a D-amino aciddehydrogenase, and a D-amino acid transaminase, thereby producingmonatin or a salt thereof.
 15. The method of claim 14, wherein at leastone of said first or second polypeptide is chosen from purifiedpolypeptides, polypeptides expressed from an exogenous microorganism,recombinant or isolated nucleic acid, chemically synthesizedpolypeptides, and polypeptides in recombinant cells or a culture ofrecombinant cells, and combinations thereof.
 16. The method of claim 14,wherein the reacting step in step (c) is performed in the presence of aninorganic base or an organic base.
 17. The method of claim 14, furthercomprising purifying said monatin or salt thereof.
 18. The method ofclaim 14, where at least about 0.058 mg/mL of monatin or salt thereof isproduced.
 19. The method of claim 14, wherein pyruvic acid or a saltthereof is contacted with said second polypeptide and indole-3-pyruvicacid or a salt thereof in step (c).
 20. The method of claim 14, furthercomprising providing an amino donor chosen from alanine, lysine, serine,glutamic acid, aspartic acid, cysteine, tryptophan, phenylalanine,methionine, tyrosine, isoleucine, leucine, valine, arginine, asparagine,glutamine, ornithine, histidine, and a combination thereof in step (e).21. The method of claim 14, further comprising purifying saidindole-3-pyruvic acid or a salt thereof prior to said reacting in step(c).
 22. A process for producing monatin or a salt thereof comprising:(a) providing tryptophan or salt thereof, (b) contacting tryptophan orsalt thereof with a first polypeptide that facilitates convertingtryptophan or salt thereof to indole-3-pyruvic acid or a salt thereof,wherein said first polypeptide is an aspartate transaminase; (c)contacting said indole-3-pyruvic acid or a salt thereof with a secondpolypeptide that facilitates converting indole-3-pyruvic acid or a saltthereof and oxaloacetic acid or a salt thereof or pyruvic acid or a saltthereof to 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a saltthereof, wherein said second polypeptide is4-hydroxy-4-methyl-2-oxoglutarate aldolase; (d) contacting said4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof witha third polypeptide that facilitates converting4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof tomonatin or salt thereof, wherein said third polypeptide is selected fromthe group consisting of an aspartate transaminase and a D-alaninetransaminase, thereby producing monatin or a salt thereof.
 23. Themethod of claim 22, wherein at least one of said first, second or thirdpolypeptide is chosen from purified polypeptides, polypeptides expressedfrom an exogenous microorganism, recombinant or isolated nucleic acid,chemically synthesized polypeptides, and polypeptides in recombinantcells or a culture of recombinant cells, and combinations thereof. 24.The method of claim 23, further comprising purifying said monatin orsalt thereof.
 25. A method for producing monatin or salt thereofcomprising contacting an aspartate transaminase with tryptophan or4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof tofacilitate a conversion of tryptophan to indole-3-pyruvate, a conversionof 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereofto monatin or a salt thereof, and a combination thereof.
 26. A methodfor producing monatin or salt thereof comprising contacting a valinetransaminase, a leucine transaminase, or a isoleucine transaminase with4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof tofacilitate a conversion of 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaricacid or a salt thereof to monatin or a salt thereof.
 27. A method forproducing monatin comprising combining, under conditions in whichmonatin is produced: (a) 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaricacid or a salt thereof; and (b) a polypeptide chosen from an aspartatetransaminase, a valine transaminase, a leucine transaminase, and aisoleucine transaminase, and a combination thereof.
 28. The method ofclaim 27, further comprising combining: (c) indole-3-pyruvic acid or asalt thereof; and (d) a second polypeptide, wherein said secondpolypeptide is capable of converting indole-3-pyruvate and oxaloaceticacid or a salt thereof or pyruvic acid or a salt thereof to4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof,wherein said second polypeptide is a 4-hydroxy-2-oxoglutarate aldolase.29. The method of claim 27, wherein said aspartate transaminase isprovided as an unpurified culture of recombinant cells.
 30. A method forproducing monatin, or a salt thereof, comprising reacting4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof andone or more D-transaminase obtained from a microorganism belonging fromthe genus Bacillus, wherein 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaricacid or a salt thereof is converted to monatin or a salt thereof. 31.The method of claim 30, wherein said microorganism belonging from thegenus Bacillus is selected from the group consisting of Bacillussphericus and Bacillus macerans.
 32. A method, comprising: producingmonatin or salt thereof via a pathway comprising producingindole-3-pyruvatic acid or a salt thereof from L-tryptophan, producing4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereofincluding at least R-isomer from4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof, andproducing the monatin from the4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof,wherein: the monatin includes at least R,R-monatin; the production ofthe 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereoffrom the L-tryptophan is facilitated by one or more first enzymes havinggreater activity, greater specificity, or both, for L-tryptophan thanfor the monatin precursor, the monatin, or both, said one or more firstenzymes are chosen from an tryptophan transaminase, a tyrosinetransaminase, an aspartate transaminase, and an L-amino aciddehydrogenase, and combinations thereof; and wherein said R,R monatinproduction from said R-isomer of4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof isfacilitated by one or more third enzymes chosen from a D-amino aciddehydrogenase and a D-amino acid transaminase or combinations thereof.33. The method of claim 32, wherein said one or more first enzymes havelimited activity, limited specificity, or both for the monatinprecursor, the monatin, or both, and optionally said one or more secondenzymes have limited activity, limited specificity, or both for theS-alpha-keto acid monatin.
 34. The method of claim 32, wherein saidD-transaminase is obtained from a microorganism belonging from the genusBacillus.
 35. The method of claim 34, wherein said microorganismbelonging from the genus Bacillus is selected from the group consistingof Bacillus sphericus and Bacillus macerans.
 36. The method of claim 32,wherein said R-isomer of 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaricacid or a salt thereof is produced from indole-3-pyruvic acid or a saltthereof in a chemical aldol condensation reaction.
 37. The method ofclaim 32, wherein the enzymes are immobilized onto a solid support. 38.A method, comprising: producing R,R monatin from a4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereofcomposition using one or more first enzyme chosen from a D-amino aciddehydrogenase and a D-amino acid transaminase, said4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereofcomposition comprising at least an R-isomer of4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof andsaid one or more enzymes selectively facilitate the conversion of theR-isomer of 4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a saltthereof over the S-isomer of4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof tomonatin.
 39. The method of claim 38, wherein said D-transaminase isobtained from a microorganism belonging from the genus Bacillus.
 40. Themethod of claim 39, wherein said microorganism belonging from the genusBacillus is selected from the group consisting of Bacillus sphericus andBacillus macerans.
 41. The method of claim 39, wherein the methodfurther comprises producing indole-3-pyruvic acid or a salt thereof fromL-tryptophan, and producing the4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereofcomposition from the indole-3-pyruvic acid or a salt thereof, wherein:the production of the indole-3-pyruvic acid or a salt thereof from the Ltryptophan is facilitated by one or more second enzymes chosen from anL-tryptophan transaminase, a tyrosine transaminase, an L-aspartatetransaminase, and an L-amino acid dehydrogenase, and combinationsthereof; and the production of the4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereofcomposition from indole-3-pyruvic acid or a salt thereof is facilitatedby one or more aldolases.
 42. A method, comprising: reacting4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof inthe presence of one or more D-transaminase to produce monatin, whereinthe one or more D-transaminase obtained from a microorganism belongingfrom the genus Bacillus.
 43. The method of claim 42, wherein saidmicroorganism belonging from the genus Bacillus is selected from thegroup consisting of Bacillus sphericus and Bacillus macerans.
 44. Amethod for producing monatin, or a salt thereof, comprising reacting4-(indol-3-ylmethyl)-4-hydroxy-2-oxoglutaric acid or a salt thereof inthe presence of one or more D-transaminase to produce monatin, whereinthe one or more D-transaminase obtained from a microorganism belongingfrom the genus Bacillus.
 45. The method of claim 44, wherein saidmicroorganism belonging from the genus Bacillus is selected from thegroup consisting of Bacillus sphericus and Bacillus macerans.